3 Live-cell Imaging of Filamentous

Fungi Using Vital Fluorescent

Dyes and Confocal Microscopy
Patrick C Hickey, Samuel R Swift, M Gabriela Roca
and Nick D Read

Live-cell Imaging of
Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh,

Filamentous Fungi
Rutherford Building, Edinburgh EH9 3JH, UK

VVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVVV

CONTENTS
Introduction
Microscope technologies for imaging living fungal cells at high spatial resolution
Vital fluorescent dyes for imaging filamentous fungi
Practical aspects of live-cell imaging
Future directions for live-cell imaging of filamentous fungi

VVVVVV INTRODUCTION

A revolutionary new perspective of the cell biology of fungal hyphae is

arising as a result of using live-cell imaging techniques to analyse
organelle and molecular dynamics at high spatial resolution. This has
become possible because of the development of a wide range of
fluorescent probes (vital dyes and fluorescent proteins) that can be used
to non-invasively interrogate living cells, new microscope technologies
(e.g. confocal and two-photon microscopy), and powerful computer
software and hardware for digital image processing and analysis. These
innovations are having a profound impact on the experimental analysis of
living fungal hyphae at the single cell level.
The aims of this review are to: (a) provide a brief comparative overview
of microscope technologies for imaging living fungal cells at high spatial
resolution; (b) review the vital fluorescent dyes which are proving useful
for analysing the cell biology of filamentous fungi with the confocal laser
scanning microscope (CLSM); (c) define important practical aspects that
need to be taken into account for optimal live-cell imaging at high spatial
resolution with the CLSM; and (d) briefly indicate future directions for
live-cell imaging of filamentous fungi.
METHODS IN MICROBIOLOGY, VOLUME 34 Copyright q 2005 Elsevier B.V.
0580-9517 DOI:10.1016/S0580-9517(04)34003-1 All rights reserved.
VVVVVV MICROSCOPE TECHNOLOGIES FOR IMAGING
LIVING FUNGAL CELLS AT HIGH SPATIAL
RESOLUTION
A range of microscope technologies have been developed over the last 20
years which allow one to image living cells at high spatial resolution using
optical sectioning. These technologies include: confocal laser scanning
microscopy (Czymmek et al., 1994; Pawley, 1995; Sheppard and Shotton,
1997; Diaspro, 2001), spinning disk confocal microscopy (Ichiwara et al.,
1999), two-photon microscopy (König, 2000; Diaspro, 2001; Feijo and

Live-cell Imaging of
Filamentous Fungi
Moreno, 2004), and deconvolution microscopy (Swedlow, 2003). The
advantages and disadvantages of these optical sectioning methods for
imaging living fungal cells are summarized in Table 3.1. Of these types of
microscopy, confocal laser scanning microscopy has been the most
popular, and most of this review is focused on results obtained using this
extremely powerful imaging technique.

VVVVVV VITAL FLUORESCENT DYES FOR IMAGING

FILAMENTOUS FUNGI
In Table 3.2 we have compiled a list of vital dyes with their selectivities
and associated parameters that we have found to be most useful for live-
cell imaging. Below we discuss the use of these dyes for studies on
filamentous fungi. We have restricted this discussion mostly to dyes
which we have personal experience of.

General Membrane-selective Dyes

The FM-dyes (FM4-64 and FM1-43) are membrane-selective probes which
stain the plasma membrane and most organelle membranes in fungal cells
(Fischer-Parton et al., 2000; Read and Hickey, 2001). They belong to a class
of amphiphilic styryl dyes developed by Betz and colleagues (Betz et al.,
1992; 1996; Bolte et al., 2004). The FM-dyes only fluoresce strongly when
located in a hydrophobic environment (e.g. a membrane) and thus,
advantageously, do not need to be washed out from the medium in which
the fungi are growing in.
The FM-dyes enter fungal cells primarily by endocytosis and thus have
been much used as endocytosis markers (see Endocytosis Marker Dyes).
Once internalized, the dyes become distributed to different organelle
membranes and different organelles stain up in a time-dependent manner
(Fischer-Parton et al., 2000; Read and Hickey, 2001; Atkinson et al., 2002;
Dijksterhuis, 2003). Much of this intracellular movement of dye seems to
be via the vesicle trafficking network, and through physical continuity
between different membranes of the endomembrane system, but other
mechanisms of dye distribution may also possibly exist (Fischer-Parton
et al., 2000). The precise pattern of organelle staining with FM4-64 is
different to that obtained with FM1-43. Both dyes can be used as general

64
 Table 3.1. Comparison of confocal laser scanning microscopy, spinning disc confocal microscopy and two-photon laser scanning microscopy for
live-cell imaging of filamentous fungi at high spatial resolution
Point of comparison Confocal laser scanning Spinning disc confocal Two-photon laser scanning Deconvolution
microscopy microscopy microscopy microscopy
Spatial resolution Very high Very high High Very high
Temporal resolution Typically 1 image/s Typically , 8 image/s Typically 1 image/s Typically , 10 image/s
65

Phototoxicity Usually most phototoxic Reduced Often reduced Reduced

Photobleaching Usually causes most Reduced Often reduced Often reduced
photobleaching
Depth of imaging Typically , 20 nm Typically , 20 nm Typically , 100 nm Typically , 20 nm
Excitation wavelengths 405 –647 nm 405 –647 nm 700 –1050 nm (two- 350 –650 nm
photon excitation)
Ease of use More difficult Relatively easy Most difficult because Relatively easy
of complex
laser system
Size Medium Medium Large Medium
Cost Expensive Expensive Most expensive Expensive

Live-cell Imaging of
Filamentous Fungi
 Table 3.2. Useful vital dyes for live-cell imaging in fungal hyphae using confocal laser scanning microscopy
Dye Selectivity Recommended excitation Maximum or Recommended dye
wavelength (nm) recommended emission concentration (mM)
wavelength(s) (nm)
Calcofluor White M2R Cell walls 405 500 0.1 –25
Carboxy DFFDA Vacuoles 488 529 10
Carboxy SNARF-1-AM pH 514 580 2 –5
FITC-dextran (10 kDa) Fluid phase endocytosis 488 514 400 mM
Carboxy SNARF-1- pH 514 580 10 mg/ml
dextran (10 kDa)
66

DASPMI Mitochondria 488 605 10 –50

DIOC6(3) Membranes 488 501 0.5 –50
Fluorescein diacetate/ Live/dead cells 488 517/617 1.5 mM/0.15 mM
propidium iodide
FM1-43 Membranes, 514 550 2.5 –25
endocytosis
FM4-64 Membranes, 514 670 2.5 –25
endocytosis
Lucifer yellow Fluid phase endocytosis 457 536 440
carbohydrazide
MDY-64 Vacuole membranes 488 497 10
Mitotracker Green Mitochondria 488 516 10
Rhodamine 123 Mitochondria 514 529 10 –65
Oregon Green-1 dextran Free calcium 488/568 530/578 1 mM/1 mM
(10 kDa)/rhodamine
B dextran (10 kDa)

Live-cell Imaging of
Filamentous Fungi
membrane-selective stains, especially for imaging cells at low magnifi-
cation (Figures 3.1, 3.2 and 3.3A; Hickey et al., 2002; Hickey and Read,
2003). For higher magnification work, FM4-64 has proved superior for
staining vacuolar membranes (Figure 3.7B,C) and vesicles within the
Spitzenkörper (Figures 3.3B, 3.4, 3.9C, and 3.12), whilst FM1-43 is best for
staining mitochondria (Figure 3.5B; Fischer-Parton et al., 2000; see
Mitochondrial Dyes). Both dyes tend to strongly stain the plasma
membrane (Figures 3.IB, 3.3A, 3.5, 3.7C and 3.9), which can be useful
for imaging septum development (Figures 3.1 and 3.5), but neither dye
stains the nuclear envelope (Figures 3.3B and 3.4B).

Live-cell Imaging of
Although many organelles become labelled with FM4-64 and FM1-43,

Filamentous Fungi
the identity of only a few are known. Furthermore, the time after which
different organelles become stained depends particularly on the cell type
and growth conditions used. Generally the staining of wide, fast growing

Figure 3.1. Neurospora crassa hyphae in the peripheral region of the colony stained with
FM1-43. (B) is of the same region as (A) but was imaged 28 min later. Note pronounced
staining of septa (arrows in B). Bar ¼ 50 mm.

67
 Live-cell Imaging of
Figure 3.2. Aspergillus fumigatus stained with FM4-64.Different stages of conidiophore Filamentous Fungi
and conidium development. (A) Single optical section. Note large vacuoles in
conidiophores. (B) 3D projection of 50 £ 0.5 mm optical sections (requires viewing
with green/red stereo glasses). Bar ¼ 20 mm.

hyphae (e.g. vegetative hyphae at the colony periphery) is quicker than

that of narrow, slow growing hyphae (e.g. germ tubes) (Table 3.3). The
initial faint staining of the cytoplasm, especially close to the plasma
membrane, is assumed to result from the labelling of a “cloud” of
endocytic vesicles (Figure 3.9A). The first fluorescent organelles appear as
punctate, roughly spherical organelles and have been assumed to be
endosomes (Figure 3.9B). Thereafter vesicles, presumed to be primarily
secretory in nature, stain up within the Spitzenkörper/apical vesicle
cluster (Figure 3.9C) indicating that endocytic pathway(s) are connected
to the secretory pathway(s). Vacuole membranes are obviously labelled

68
 Live-cell Imaging of
Filamentous Fungi
Figure 3.3. Hyperbranching spray mutant of Neurospora crassa stained with FM4-64.
(A) Low-magnification image. Note multiple stained septa (arrows). (B) High-
magnification image showing irregular growth of hyphae, multiple branches (many
of which abort), small Spitzenkörper (s) and negatively stained nuclei (n).
Bar ¼ 10 mm.

and in N. crassa, the large spherical vacuoles (Figure 3.7B,C) become

stained before those of the tubular vacuolar network (Figure 3.7A; Read
and Hickey, 2001; Table 3.3). Interestingly, mitochondrial membranes also
stain up with FM4-64 and FM1-43, but most markedly with FM1-43 (see
Mitochondrial Dyes) (Fischer-Parton et al., 2000).
In future, it will be very important to identify the different organelles
stained with the FM-dyes. One approach will be to double label cells with
a FM-dye and a fluorescent protein targeted to a specific organelle.
This approach has proved particularly useful in demonstrating that Golgi,
but not ER, are stained by FM4-64 in plant cells (Bolte et al., 2004). Another
approach will be to localize the FM-dyes at the ultrastructural level, and
methods for this have been developed (Henkel et al., 1996).
After prolonged staining (. 30 min), FM4-64 and FM1-43 have proven
to be the best dyes for providing general staining of living hyphae and
sporulating structures, especially when viewed at low magnifications
(e.g. with a 20 £ objective, Figures 3.1, 3.2 and 3.3A). These dyes are
particularly excellent for characterizing the morphological phenotypes of
mutants in the living state (Figures 3.3 and 3.11).
Another membrane-selective dye which has been used in filamentous
fungi is DIOC6(3). This is a cationic, cyanine dye which accumulates in the
mitochondria, endoplasmic reticulum (ER) and nuclear envelope in a
potential-dependent manner. In plants, low dye concentrations favour
mitochondrial staining whilst high concentrations will stain ER and the

69
 Live-cell Imaging of
Filamentous Fungi
Figure 3.4. (A) Neurospora crassa hypha stained with FM4-64 showing sub-apical
branch formation. Note the initiation of the Spitzenkörper beneath the plasma
membrane (arrow) that has appeared just before the branch emerged. (B) Sclerotinia
sclerotiorum hyphae stained with FM4-64 showing apical branching. Note the
negatively stained nuclei (n) and less stained core region within the Spitzenkörper.
Bars ¼ 10 mm.

nuclear envelope (Oparka and Read, 1994). DIOC6(3) has also been used
to stain mitochondria, ER and the nuclear envelope in yeast cells (e.g.
Koning et al., 1993) and filamentous fungi (e.g. Koll et al., 2001).

Nuclear Dyes
We have not found any vital nuclear dye to be particularly good for live-
cell imaging of fungal hyphae using confocal microscopy.
DAPI is usually used to stained AT-rich regions of DNA within fixed
fungal cells. However, it can sometimes be used as a vital probe if the cell
is permeable to the dye. Often we find that vegetative hyphae will not take
up DAPI whilst germinating spores will. DAPI is normally excited at UV
wavelengths which are not very compatible with live-cell imaging.
However, it is also excited at 405 nm which is now available for confocal

70
 Live-cell Imaging of
Filamentous Fungi
Figure 3.5. Septum formation in Neurospora crassa stained with FM4-64. Only the
plasma membrane on either side of the septum is stained. Bar ¼ 10 mm.

microscopy in the form of a relatively inexpensive blue diode laser.

However, so far we have not been able to perform good live-cell nuclear
imaging with DAPI using the CLSM because of the staining of other
cellular components (e.g. mitochondrial DNA and possibly polypho-
sphates) (Roca, M. G. and Read, N. D., unpublished results).
A wide range of cyanine Syto dyes, which stain both DNA and RNA,
are available from Molecular Probes Inc. (Haugland, 2002). We have
tested a wide range of green fluorescing Syto dyes (Syto 11, 13, 14, 15
and 16) and have found them to be readily cell permeant and to stain
nuclei in vegetative hyphae to varying extents. However, these dyes have
not proven useful in our live-cell imaging studies because all are very
phototoxic and cause organelle disruption when scanned with a laser
beam under the CLSM (Hickey, 2001).
As indicated in General Membrane-selective Dyes, DIOC6(3) can stain
up the nuclear envelope, along with mitochondria and ER, if used at high

Table 3.3. Summary of the time course of FM4-64 staining at 20– 258C of
vegetative hyphae of Neurospora crassa at the colony periphery and conidial germ
tubes of Magnaporthe grisea (data from: Read and Hickey, 2001; Atkinson et al.,
2002)
Cell component stained Approximate times after adding FM4-64

Vegetative hyphae of Germ tubes of

N. crassa M. grisea
Plasma membrane Immediate Immediate
Putative endocytic vesicles , 10 s 1 –2 min
Putative endosomes 30 s 2 min
Vesicles within Spitzenkörper 1.5 min 2 min
Large spherical vacuoles 10 min 45 min
Tubular vacuoles 15 min 45 min

71
 Figure 3.6. Mitochondrial staining. (A) Aspergillus nidulans stained with Rhodamine-

Live-cell Imaging of
Filamentous Fungi
123. Note that the fluorescence is higher in the hyphal tip regions where the
mitochondria are apparently most active. Bar ¼ 20 mm. (B) Neurospora crassa hyphal tip
stained with FM1-43. (C) Sclerotinia sclerotiorum hyphal tip stained with DASPMI.
Bars ¼ 10 mm (for B and C).

concentrations (e.g. . 25– 50 mM). Nuclei can also be often imaged as

negatively stained structures in cells which have undergone prolonged
staining with FM4-64 (Figures 3.3B and 3.4B). By far the best way of
visualizing nuclei in fungi is using a nuclear-targeted fluorescent protein
(e.g. GFP) (e.g. Freitag et al., 2004a,b; Figure 3.12). For a detailed
description of the use of fluorescent proteins in live-cell imaging see
Chapter 2.

Mitochondrial Dyes
Within apical hyphal compartments, mitochondria are typically tubular
and long although the length and thickness of individual mitochondria
varies between species (Figure 3.6B,C; Fischer-Parton et al., 2000). In
actively growing vegetative hyphae the mitochondria are typically tightly
packed and numerous (Figure 3.5A – C). In sub-apical regions, mitochon-
dria are shorter and more diffusely distributed (Fischer-Parton et al.,
2000). In slow growing germ tubes mitochondria are fewer and shorter
(Swift, S., Hickey, P. C. and Read, N. D., unpublished). It is not clear to
what extent, if any, that mitochondria in filamentous fungal cells are
branched as occurs, for example, in budding yeast cells (Hermann and
Shaw, 1998). We have found the following dyes to be useful for staining
mitochondria in filamentous fungal cells: Rhodamine 123, DASPMI,
FM1-43 and Mitotracker green.
Rhodamine 123 (Johnson et al., 1980) is a cell-permeant, cationic,
fluorescent dye that is sequestered by active mitochondria of hyphae
within a few minutes. It is a potentiometric dye, the fluorescence of which
is directly dependent on the electrochemical gradient across the
mitochondrial membrane (the greater the membrane potential, the greater
the fluorescence). Generally the mitochondria close to growing tips
fluoresce more brightly than those further back indicating that those at the
tip are most active (Figure 3.6A). This may be due to the need for high
levels of ATP which are required to maintain tip growth. In Botrytis

72
 cinerea we found that Rhodamine 123 fluorescence could be quenched
by collapsing the electrochemical gradient of mitochondrial membranes
by treatment with the fungicide azoxystrobin which inhibits mitochon-
drial membrane transport at the cytochrome bc1 complex (Swift et al., 2001).
Rhodamine 123 also stains the hyphal plasma membrane but
often gives background staining of the medium (Figure 3.5C; Fischer-
Parton et al., 2000).
DASPMI is a styryl dye (Bereiter-Hahn, 1976) and takes much longer to
stain mitochondria (, 20 min) than does Rhodamine 123. It is also a
potentiometric dye but to a much lesser extent than Rhodamine 123. It
does not stain the plasma membrane or give background staining of the

Live-cell Imaging of
Filamentous Fungi
medium. A useful feature of DASPMI is that it exhibits a large Stoke’s
shift with blue excitation and red emission (Table 3.2). It is thus well
suited in dual labelling studies (e.g. with GFP which is excited with blue
light and emits green fluorescence, Freitag et al., 2004b).
The FM-dyes were developed from DASPMI (Bolte et al., 2004). FM1-43
stains mitochondria of N. crassa more rapidly (after ,20 min) compared
with FM4-64 (after ,45 min). FM1-43 is also much more selective for
mitochondria and stains them more intensely than does FM4-64 (Figure
3.5A; Fischer-Parton et al., 2000) and thus is the preferred mitochondrial stain
of the two dyes. FM1-43 also stains the plasma membrane (Figure 3.5A) but
to a lesser extent than does Rhodamine 123 (Figure 3.5C). It is not clear how
mitochondria are stained by these dyes (i.e. by stained vesicles fusing with
mitochondria, by stained membrane being in contact with mitochondria, or
possibly by some other mechanism (Fischer-Parton et al., 2000)).
MitoTracker probes are cell-permeant mitochondrion-selective dyes
that passively diffuse across the plasma membrane and accumulate in
active mitochondria. Various Mitotracker probes are commercially avail-
able from Molecular Probes Inc. (Haugland, 2002). We have found that
Mitotracker Green stains mitochondria in hyphae well (data not shown).
DIOC6(3) will stain mitochondria in hyphae and spores when used at
low concentrations (typically , 0.5 mM) but we have been unable to
obtain mitochondrial staining as good as we routinely achieve with the
other mitochondrial dyes.

Vacuolar Dyes
The vacuolar system of filamentous fungi is very complex being
composed of a network of very dynamic tubular elements in young
hyphal compartments (Figure 3.7A) and large spherical organelles in
older compartments (Figures 3.2, 3.7B – D), as well as many small
spherical organelles (Figures 3.7A,B) (Cole et al., 1998; Ashford et al.,
2001). Three dyes which are useful for staining the vacuolar system are
FM4-64, MDY-64 and DFFDA.
FM4-64 (see General Membrane-selective Dyes and Endocytosis
Marker Dyes) stains vacuolar membranes as the end point of the
endocytic pathway (Figures 3.2 and 3.7C). In Neurospora vegetative
hyphae FM4-64 starts to stain the large spherical vacuoles after , 10 min

73
 Live-cell Imaging of
Filamentous Fungi
Figure 3.7. Vacuole staining in Neurospora crassa. (A) Tubular vacuolar network in
apical hyphal compartment and branch stained with carboxy DFFDA. (B) Large and
small spherical vacuoles in sub-apical hyphal compartment stained with carboxy-
DFFDA. (C) Vacuole membrane of large spherical vacuole in sub-apical hyphal
compartment stained with FM4-64. (D) Vacuole membranes within conidia of
Colletotrichum lindemuthianum stained with MDY-64. Note that the two conidia are
fusing via conidial anastomosis tubes (arrow). All bars ¼ 10 mm.

and tubular elements after , 20 min (Table 3.3; Read and Hickey, 2001). In
Magnaporthe germ tubes, vacuolar compartments stain up later (, 45 min,
Table 3.3; Atkinson et al., 2002).
MDY-64 is also a yeast vacuolar dye which has been found to also stain
up the membranes of vacuoles, and possibly other membranes in spores
and hyphae (Figure 3.7D; Cole et al., 1998; Roca et al., 2003), although the
precise mechanism of staining is not known.
Oregon Green 488 carboxylic acid (carboxy-DFFDA) is a derivative of
6-carboxyfluorescein diacetate (CFDA) but more photostable than it. Cole
et al. (1997) provided evidence that both of these lipophilic probes cross
the plasma membrane of hyphae of Pisolithus tinctorius, and are
hydrolysed in the cytoplasm to Oregon Green 488 carboxylic acid and
6-carboxyfluorescein, respectively. They then seem to be rapidly trans-
ported across the vacuolar membrane into vacuoles by an organic anion
transporter as this step can be inhibited by the anion transport inhibitor
probenecid. Carboxy-DFFDA is an excellent dye for staining the lumens
of both tubular and spherical vacuoles within hyphae (Figure 3.6A,B).

74
 Live-cell Imaging of
Filamentous Fungi
Figure 3.8. Growth of a hyphal branch of Neurospora crassa stained with 0.1 mM
Calcofluor White M2R. Bar ¼ 5 mm.

Cell Wall Dyes

Calcofluor White M2R is a cell wall stain that binds to chitin by
intercalating into nascent chitin chains, and this prevents chitin
microfibril assembly (Elorza et al., 1983). It is a widely used cell wall
stain for yeast and hyphal cells (both fixed and living) but is normally
used at concentrations (typically . 25 mM) that increase chitin synthesis
and interfere with cell growth (Roncero and Duran, 1985). Nevertheless,
at very low concentrations (, 1.5 mM), Calcofluor can be useful as a live-
cell stain (Figure 3.8).

Endocytosis Marker Dyes

It is generally believed that the FM-dyes are excellent endocytosis
markers and reporters of vesicle trafficking in animal, plant, yeast and
filamentous fungal cells (Vida and Emr, 1995; Cochilla et al., 1999;
Fischer-Parton et al., 2000; Bolte et al., 2004) although there has been some
debate about whether these dyes might also be internalized and
transported around cells by other mechanisms as well (Fischer-Parton
et al., 2000; Torralba and Heath, 2002; Bolte et al., 2004).
The FM-dyes are amphiphilic molecules which are believed to get
inserted in the outer leaflet of the plasma membrane bilayer. Because of
their amphiphilic nature they are thought to be unable to directly cross the
plasma membrane. The primary mechanism of FM-dye entry into cells is
probably via endocytic vesicles which are invaginated from the plasma
membrane. As indicated in General Membrane-Selective Dyes, these
vesicles are then believed to be transported to endosomes, and

75
 Live-cell Imaging of
Filamentous Fungi
Figure 3.9. Time course of FM4-64 staining of a hyphal tip of Sclerotinia sclerotiorum.
Bar ¼ 10 mm.

subsequently to other organelles in the vesicle trafficking network resulting

in the staining of different organelles in a time-dependent manner (Figures
3.9A – C) (Fischer-Parton et al., 2000; Read and Hickey, 2001; Dijksterhuis,
2003). The endpoint of the endocytic pathway is lytic vacuoles and as
described in Vacuolar Dyes, the FM-dyes are good probes for staining
vacuolar membranes (Figure 3.2, 3.7C). Consistent with FM-dye uptake
being an active process involving endocytosis is the finding that this
internalization is inhibited in filamentous fungi by treatments (low temper-
ature or addition of sodium azide) which inhibit ATP production (Hoffman
and Mendgen, 1998; Fischer-Parton et al., 2000; Atkinson et al., 2002).
Other mechanisms of FM4 dye internalization (e.g. by flippases) or
transport between different organelles (e.g. by lipid transport proteins)
have been suggested but we are as yet unaware of any convincing
evidence to support the involvement of these alternative pathways
(Fischer-Parton et al., 2000; Read and Kalkman, 2003).
Another class of dyes have been used as markers of fluid-phase
endocytosis because they are commonly believed to be unable to cross the
plasma membrane by passive diffusion but can become trapped within
the lumen of endocytic vesicles invaginated from the plasma membrane
(Dulic et al., 1991; Hawes et al., 1995). These dyes include Lucifer Yellow
carbohydrazide (LYCH) and FITC-dextran. Only very limited success
with the uptake of these probes into the cells of filamentous fungi has been
achieved (Steinberg et al., 1998; Atkinson et al., 2002) and there are several
reports of these probes not being taken up by hyphae (Cole et al., 1997;
Steinberg et al., 1998; Fischer-Parton et al., 2000; Torralba and Heath, 2002).
The significance of these results is unclear (Read and Kalkman, 2003).

76
Ca21 and pH Dyes
A very large range of pH- and Ca2þ-selective dyes are commercially
available (Parton and Read, 1999; Haugland, 2002) for providing
quantitative, spatially and temporally resolved measurements of intra-
cellular Hþ or Ca2þ concentrations as they undergo dynamic changes
within living cells. Most of these probes are designed to measure pH and
free Ca2þ in the cytosol.
The use of pH- and Ca2þ-dyes for imaging and measuring ion
concentrations in filamentous fungi is fraught with problems. The first
problem is often associated with introducing the dye into the cell, and this
can be particularly difficult with Ca2þ-dyes (Read et al., 1992; Knight et al.,

Live-cell Imaging of
Filamentous Fungi
1993; Parton and Read, 1999). The second problem is that the dyes
frequently become sequestered within organelles, and when this occurs in
sub-200 nm vesicles it can often be extremely difficult to detect. This can
potentially have a profound effect in artifactually generating what appear
to be cytosolic ionic gradients. An important control to verify that data
obtained using free dyes are reliable is to check whether one obtains
similar results with either a dextran-conjugated form of the dye
(Parton et al., 1997) or a cytoplasmically targeted ion-sensitive recombi-
nant probe (Miyawaki, 2003), neither of which should be taken up by
organelles. Pressure injection of hyphae with dextran-conjugated dyes is
not easy but is facilitated by using a pressure probe (Parton et al., 1997).
A third problem is quantitation. In hyphae we have found that the only
satisfactory quantitative method is to use a ratiometric approach in order
to overcome problems of variations in fluorescence brightness due to
variations in dye concentration within a cell, cell thickness, dye leakage or
dye photobleaching (Parton and Read, 1999).
The only pH-sensitive dye we would recommend for pH imaging with
the CLSM is carboxy-SNARF-1 which is a dual emission ratiometric dye.
We have successfully used this in both its free form (loaded as its AM-
ester) and 10 kDa dextran-conjugated form (Parton et al., 1997).
Calcium ratio imaging with the CLSM is much more difficult to
perform because there are no ratiometric dyes which are very suitable
for excitation with visible light wavelengths. An alternative approach is
to simultaneously load cells with two dyes and ratio one against the
other. In our lab, Fischer-Parton (1999) used this approach successfully
by pressure injecting both the 10 kDa dextran of Oregon Green (which
is Ca2þ sensitive) with the 10 kDa dextran of Rhodamine B (which is
Ca2þ insensitive). Silverman-Gavrila and Lew (2000, 2001, 2003) have
ratio imaged Ca2þ in hyphae dual loaded with the Ca2þ-dyes Fluo3-AM
and Fura-Red-AM, although they did not perform appropriate controls
with a dextran-conjugated dye or recombinant probe as mentioned
above. The methods and problems of imaging and measuring free Ca2þ
and pH are discussed in detail by Parton and Read (1999). Hopefully,
many of the problems will be solved in the future with recombinant
probes which have been optimized for Ca2þ and pH measurement in
filamentous fungi.

77
Dyes to Test Cell Viability
It is often very useful to test the viability of fungal cells in a cell
population. We have found that combining the stains fluorescein
diacetate (FDA) and propidium iodide works extremely well for this
purpose (Oparka and Read, 1994). Both FDA and propidium iodide can
be excited at 488 nm. FDA is readily taken up by living cells and is
hydrolysed to fluoroscein which fluoresces green. Propidium iodide,
however, is only taken up by cells which are dead or have had their
plasma membrane damaged. Only dead or damaged cells, therefore,
take up propidium iodide and these fluoresce red. By simultaneously
imaging at the appropriate emission wavelengths (Table 3.3) it is thus

Live-cell Imaging of
Filamentous Fungi
possible to determine which cells are living (green) and which are dead
(red). Because fluorescein is poorly retained by cells, the live/dead cell
assay should be performed immediately after adding the dyes. An
alternative to using FDA is to use BCECF which is better retained by
cells (Haugland, 2002).

VVVVVV PRACTICAL ASPECTS OF LIVE-CELL IMAGING

Optimal live-cell imaging with the CLSM requires the use of “low-dose”
techniques (e.g. using dyes at low non-cytotoxic concentrations with
levels of laser irradiation at non-harmful visible wavelengths). Live-cell
imaging thus requires more compromises than “dead-cell imaging” (i.e.
of fixed cells). It is important to treat one’s cells with the respect that they
deserve in order that they remain as healthy as possible. The following are
important practical aspects which one needs to pay close attention whilst
imaging living fungal cells.

Sample Preparation
We have found that the simplest method for preparing samples in order
that hyphae stay in the same plane of focus (especially important when
imaging at low magnification, Figures 3.1, 3.2, 3.3A) and to reduce
spherical aberration to a minimum (and thus have sharply in focus
images) is to use the “inverted agar block method” (Figure 3.10). This
involves taking a , 20 mm2, 5 mm thick block of agar from whichever
region of the colony needs to be imaged, and inverting it onto a droplet of
liquid medium (containing dye) upon a glass cover slip (Hickey and
Read, 2003). Excess medium can be removed with a filter paper wick. If
the hyphae tend to grow into the agar, this can sometimes be inhibited by
using a higher agar concentration (e.g. 3% w/v). If using a confocal
system mounted on an inverted microscope then it is also useful to place a
Petri dish containing water-soaked filter paper over the sample to reduce
desiccation to a minimum. Most of the figures shown in the paper were
from samples prepared in this way. Using the inverted agar block method
we are usually able to image samples for 1 h or more without apparent
deleterious effects on growing hyphae (e.g. from O2 deprivation).

78
 Live-cell Imaging of
Filamentous Fungi
Figure 3.10. Inverted agar block culture method.

Temperature-sensitive mutants can be readily imaged at their

restrictive temperatures (normally between 35 and 428C), or during
temperature shift down or shift up, by using a temperature-controlled
stage or temperature-controlled box around the confocal microscope.
However, it should be noted that rates of evaporation from the sample
will be increased at elevated temperatures and thus extra liquid medium
may have to be added more frequently during imaging. In Figure 3.11 we
show an example of 4D imaging (x; y; z and time) with the cot-1 mutant of
Neurospora during a temperature shift down from the non-permissive
temperature (378C) to the permissive temperature (258C). This tempera-
ture decrease results in the hyperbranching phenotype of the mutant
(shown in yellow) reverting to the normal hyphal morphology of the wild
type (shown in red).

Multiple Labelling Living Cells

Multiple labelling is often possible with living fungal cells. We routinely
combine the red fluorescing FM4-64 as a general membrane stain with
GFP targeted to different organelles or fused to different proteins
(Figure 3.12; Freitag et al., 2004b). The red fluorescing mitochondrial
stain DASPMI can also be used in combination with green fluorescing
probes such as GFP (Freitag et al., 2004b). Triple labelling is also possible
(e.g. with Calcofluor, GFP and FM4-64. Certain combinations of dyes,
however, are sometimes incompatible and can result in increased
phototoxicity when imaged with the CLSM.

The Signal-to-noise Ratio and Spatial

versus Temporal Resolution
In the context of this chapter, the signal-to-noise ratio (S/N) is the ratio
of the fluorescent signal to the noise from the surrounding background

79
 Live-cell Imaging of
Filamentous Fungi

Figure 3.11. Hyperbranching cot-1 mutant of Neurospora crassa stained with FM4-64
grown at 378C for 3 h and shifted back to 258C. Each image represents a projection of
30 £ 1.0 mm optical sections through the hypha. The times represent the periods after
which the hyphae were shifted to 258C. Note the bulbous compartments of the main
hypha and the finely tapered branches (yellow-green) which become wider during
recovery of the wild type hyphal phenotype (red). Bar ¼ 20 mm.

80
 Live-cell Imaging of
Filamentous Fungi
Figure 3.12. Morchella esculenta. Hyphal tip stained with FM4-64. Note retraction of the
Spitzenkörper and formation of a new one after 24 s. Bar ¼ 10 mm.

(e.g. autofluorescence and electronic noise from the photomultiplier

detectors) from which the signal must be distinguished (for a detailed
account of noise the reader is referred to Murphy (2001)). Ways
of improving the S/N ratio with accompanying problems are indicated
in Table 3.4.
Another problem is the compromise that needs to be made between
spatial and temporal resolution. Generally increasing the temporal
resolution (and thus the time period over which each image is
captured) reduces the S/N and the spatial resolution achieved. The
S/N ratio can be increased in various ways (see Table 3.4) but this
usually results in increased cytotoxicity, phototoxicity, and/or photo-
bleaching. Therefore, the live-cell imager needs to pay much closer
attention to the microscope parameters that he/she is using than does
the dead-cell imager.

Refractive Index Problems

Multiple changes in the refractive index between the objective and cell
and within the cell will significantly reduce the spatial resolution and

81
 Table 3.4. Ways of improving the signal-to-noise ratio for confocal laser
scanning microscopy, and the potential problems caused
Ways of increase S/N Potential problems
ratio
Use dyes with a –
high quantum yield, which
are not phototoxic and
which are resistant to
photobleaching
Optimize excitation and emission –
wavelengths

Live-cell Imaging of
Filamentous Fungi
Use high NA objectives Increased phototoxicity
Increased photobleaching
Increase dye concentration Increased cytotoxicity
Increased phototoxicity
Increase laser power Increased phototoxicity
Increased photobleaching
Increase electronic zoom during Increased phototoxicity
imaging Increased photobleaching
Decrease rate of laser Increased phototoxicity
scanning Increased photobleaching
Integrate or average successive Increased phototoxicity
images Increased photobleaching
Open pinhole Reduced spatial resolution

cause spherical aberration (Murphy, 2001). Some relevant refractive

indices here are: air (1.0), immersion oil (1.52), cover slip glass (1.52),
growth medium (. 1.33), and cytoplasm (. 1.33 but varies in different
parts of a cell). These refractive index problems (and thus spherical
aberration) are accentuated the further the cell is away from the cover slip.
Ways of reducing these problems are to: reduce the number of changes of
refractive index between the objective and the cell, use oil immersion,
water immersion or dipping objectives (without a cover slip), and keep
cells as close as possible to the cover slip.

Indicators of Cell Perturbation During Live-cell Imaging

We have found that hyphae of different species loaded with dyes vary in
their sensitivities to being scanned with lasers of the CLSM, and varying
degrees of cytotoxicity and phototoxicity are experienced with different
fluorescent probes and at different excitation wavelengths. It is, therefore,
important to pay close attention to the health of one’s sample. Various
indicators of cell perturbation which can be readily assessed are: slowing
down of hyphal growth; narrowing down of the hyphal tip, retraction of
the Spitzenkörper from the hyphal tip (Figure 3.12), and changes in
organelle morphology.

82
Movie-making
One of the major advantages of live-cell imaging is that one is able to
perform time-lapse experiments and make movies of time-lapse imaging.
This can provide an extraordinary insight into the dynamics of labelled
organelles and molecules in living fungal cells. Typically we capture
50 –500 images at 2 – 30 s intervals in a time course. These images may
then be processed (e.g. contrast and brightness adjusted, cropped and
merged), and imported into a movie-making software package (e.g.
Adobe Premiere) in which they can be compressed, edited and labelled.
We normally save the final movie files in an avi or mpg format which can
be easily inserted into Microsoft Powerpoint for presentation purposes.
Examples of movies of filamentous fungi imaged with the CLSM after

Live-cell Imaging of
Filamentous Fungi
staining with many of the dyes described in this chapter can be found on a
CD-ROM (Hickey and Read, 2003) which we have produced for
educational purposes, and can be obtained from www.fungalcell.org.

VVVVVV FUTURE DIRECTIONS FOR LIVE-CELL IMAGING

OF FILAMENTOUS FUNGI
In this chapter, we have restricted ourselves to discussing those dyes
which are well tried and tested in our lab. Many other commercially
available dyes will undoubtedly prove extremely useful for live-cell
imaging of filamentous fungi, and the experimenter is strongly
encouraged to experiment with them. A good starting point is to leaf
through the Molecular Probes catalogue (Haugland, 2002) and explore the
Molecular Probes website (www.probes.com). We can expect the number
of dyes for live-cell imaging to continually increase and these will
compliment the rapidly increasing range of recombinant fluorescent
probes which are also being developed (Zhang et al., 2002).
In this chapter, we have also restricted ourselves to discussing the
imaging of filamentous fungi with the CLSM because this is the optical
sectioning technology which is most widely used and is the one with
which we have most experience. However, other optical sectioning
techniques (Table 3.1) have different advantages (and disadvantages) to
confocal laser scanning microscopy and for certain live-cell imaging
applications will provide superior or complimentary results. Further-
more, low-light wide-field fluorescence imaging should also be routinely
employed, especially for detecting very low levels of fluorescence. In
addition, there are other important technological developments which are
beginning to have an impact on live-cell imaging and analysis in
filamentous fungi. These include: fluorescence recovery after photo-
bleaching (FRAP) (Lippincott-Schwartz et al., 2001), spectral imaging
(Berg, 2004), fluorescence resonance energy transfer (FRET) imaging
(Periasamy and Day, 1999; Kenworthy, 2001), fluorescence lifetime
imaging microscopy (FLIM) (Lacowicz, 1997; Gadella, 1999) and
fluorescence correlation spectroscopy (FCS) (Medina and Schwille, 2002;
Levin and Carson, 2004).

83
Acknowledgements
Thanks are due to the Biological and Biotechnological Sciences Research
Council for a CASE Studentship to SRS, a Research Studentship to PCH
and a grant (15/P18594) to NDR. Part of the research involved the use of
CLSM equipment in the COSMIC (Collaborative, Optical, Spectroscopy,
Micromanipulation and Imaging Centre) facility which is a Nikon
Partners-in-Research laboratory at the University of Edinburgh.

References
Ashford, A. E., Cole, L. and Hyde, G. J. (2001). Motile tubular vacuole systems. In
Biology of the Fungal Cell., The Mycota (R. J. Howard and N. A. R. Gow, eds),

Live-cell Imaging of
Filamentous Fungi
vol. VIII, pp. 243– 265. Springer, Berlin.
Atkinson, H. A., Daniels, A. and Read, N. D. (2002). Live-cell imaging of
endocytosis during conidial germination in the rice blast fungus, Magnaporthe
grisea. Fungal Genet. Biol. 37, 233– 244.
Bereiter-Hahn, J. (1976). Dimethylaminostyrylmethylpyridiniumiodine (daspmi)
as a fluorescent probe for mitochondria in situ. Biochim. Biophys. Acta 423, 1 – 14.
Berg, R. H. (2004). Evaluation of spectral imaging for plant cell analysis. J. Microsc.
214, 174– 181.
Betz, W. J., Mao, F. and Bewick, G. S. (1992). Activity-dependent fluorescent
staining and destaining of living vertebrate motor nerve terminals. J. Neurosci.
12, 363– 375.
Betz, W. J., Mao, F. and Smith, C. B. (1996). Imaging exocytosis and endocytosis.
Curr. Opin. Neurobiol. 6, 365– 371.
Bolte, S., Talbot, C., Boutte, Y., Catrice, O., Read, N. D. and Satiat-Jeunemaitre, B.
(2004). FM-dyes as experimental probes for dissecting vesicle trafficking in
living plant cells. J. Microsc. 214, 159– 173.
Chen, L. B. (1989). Fluorescent labeling of mitochondria. Meth. Cell Biol. 29,
103–123.
Cochilla, A. J., Angleson, J. K. and Betz, W. J. (1999). Monitoring secretory
membrane with FM1-43 fluorescence. Annu. Rev. Neurosci. 22, 1 –10.
Cole, L., Hyde, G. J. and Ashford, A. E. (1997). Uptake and compartmentalisation
of fluorescent probes by Pisolithus tinctorius hyphae: evidence for an anion
transport mechanism at the tonoplast but not for fluid-phase endocytosis.
Protoplasma 199, 18 – 29.
Cole, L., Orlovich, D. A. and Ashford, A. E. (1998). Structure, function and motility
of vacuoles in filamentous fungi. Fungal Genet. Biol. 24, 86 – 100.
Czymmek, K. J., Whallon, J. H. and Klomparens, A. (1994). Confocal microscopy in
mycological research. Exp. Mycol. 18, 275– 293.
Czymmek, K. J., Bourett, T. M., Sweigard, J. A., Carrol, A. and Howard, R. J. (2002).
Utility of cytoplasmic fluorescent proteins for live-cell imaging of Magnaporthe
grisea in planta. Mycologia 94, 280– 289.
Diaspro, A. (2001). Confocal and Two-Photon Microscopy: Foundations, Applications,
and Advances. Wiley-Liss, New York.
Dijksterhuis, J. (2003). Confocal microscopy of Spitzenkörper dynamics during
growth and differentiation of rust fungi. Protoplasma 222, 53 – 59.
Dulic, V., Egerton, M., Elgundi, I., Raths, S., Singer, B. and Riezman, H. (1991).
Yeast endocytosis assays. Meth. Enzymol. 194, 697– 710.
Elorza, M. V., Rico, H. and Sentandreu, R. (1983). Calcofluor white alters the
assembly of chitin fibrils in Saccharomyces cerevisiae and Candida albicans cells.
J. Gen. Microbiol. 129, 1577– 1582.

84
Feijo, J. A. and Moreno, N. (2004). Imaging plant cells by two-photon excitation.
Protoplasma 223, 1 –32.
Fischer-Parton, S. (1999). Role of pH, calcium and vesicle trafficking in regulating
tip growth of Neurospora crassa. PhD Thesis. University of Edinburgh,
Edinburgh, UK.
Fischer-Parton, S., Parton, R. M., Hickey, P. C., Dijksterhuis, J., Atkinson, H. A. and
Read, N. D. (2000). Confocal microscopy of FM4-64 as a tool for analysing
endocytosis and vesicle trafficking in living fungal hyphae. J. Microsc. 198,
246–259.
Freitag, M., Hickey, P. C., Khlafallah, T. K., Read, N. D. and Selker, E. U. (2004a).
HP1 is essential for DNA methylation in Neurospora. Mol. Cell. 13, 427– 434.
Freitag, M., Hickey, P. C., Raju, N. B., Selker, E. U. and Read, N. D. (2004b). GFP as a
tool to analyze the organization, dynamics and function of nuclei and

Live-cell Imaging of
microtubles in Neurospora crassa. In Press.

Filamentous Fungi
Gadella, T. W. J. (1999). Fluorescence lifetime imaging microscopy (FLIM):
instrumentation and applications. In Fluorescent and Luminescent Probes for Biological
Activity (W. T. Mason, ed.), 2nd edn., pp. 467–479. Academic Press, London.
Haugland, R. P. (2002). Handbook of Fluorescent Probes and Research Products, 9th
edn. Molecular Probes Inc., Eugene, OR.
Hawes, C., Crooks, K., Coeman, J. and Satiat-Jeunemaitre, B. (1995). Endocytosis in
plants: fact or artefact? Plant Cell Environ. 18, 1245 –1252.
Henkel, A., Lubke, J. and Betz, W. (1996). FM1-43 dyes ultrastructural localization
in and release from frog motor nerve terminals. Proc. Natl Acad. Sci. USA 93,
1918–1923.
Hermann, G. J. and Shaw, J. M. (1998). Mitochondrial dynamics in yeast. Annu.
Rev. Cell Dev. Biol. 14, 265– 303.
Hickey, P. C. (2001). Imaging vesicle trafficking and organelle dynamics in living
fungal hyphae. PhD Thesis. University of Edinburgh, Edinburgh, UK.
Hickey, P. C. and Read, N. D. (2003). Biology of Living Fungi. British Mycological
Society, Stevenage, UK, CD-ROM.
Hickey, P. C., Jacobson, D. J., Read, N. D. and Glass, N. L. (2002). Live-cell imaging
of vegetative hyphal fusion in Neurospora crassa. Fungal Genet. Biol. 37, 109– 119.
Hoffman, J. and Mendgen, K. (1998). Endocytosis and membrane turnover in the
germ tube of Uromyces fabae. Fungal Genet. Biol. 24, 77 – 85.
Ichiwara, A., Tanaami, T., Ishida, H. and Shimizu, M. (1999). Confocal fluorescent
microscopy using a Nipkow scanner. In Fluorescent and Luminescent Probes for
Biological Activity (W. T. Mason, ed.), 2nd edn., pp. 344– 349. Academic Press,
San Diego.
Johnson, L. V., Walsh, M. L. and Chen, L. B. (1980). Localization of mitochondria in
living cells with rhodamine 123. Proc. Natl Acad. Sci. USA 77, 990– 994.
Kenworthy, A. K. (2001). Imaging protein – protein interactions using fluorescence
resonance energy transfer microscopy. Methods 24, 289– 296.
Knight, H., Trewavas, A. J. and Read, N. D. (1993). Confocal microscopy of living
fungal hyphae microinjected with Ca2þ-sensitive fluorescent dyes. Mycol. Res.
97, 1505– 1515.
Koll, F., Sidoti, C., Rincheval, V. and Lecellier, G. (2001). Mitochondrial membrane
potential and ageing in Podospora anserina. Mech. Ageing Dev. 122, 205–217.
König, K. (2000). Multiphoton microscopy in life sciences. J. Microsc. 200, 83 – 104.
Koning, A. J., Lum, P. Y., Williams, J. M. and Wright, R. (1993). DIOC6 staining
reveals organelle structure and dynamics in living yeast cells. Cell Motil.
Cytoskel. 25, 111 – 128.
Lacowicz, J. R. (1997). Topics in Fluorescence Spectroscopy: Nonlinear and Two-Photon-
Induced Fluorescence, vol. 5. Plenum Press, New York.

85
Levin, M. K. and Carson, J. H. (2004). Fluorescence correlation spectroscopy and
quantitative cell biology. Differentiation 72, 1 – 10.
Lippincott-Schwartz, J., Snapp, E. and Kenworthy, A. (2001). Studying protein
dynamics in living cells. Nat. Rev. Mol. Cell Biol. 2, 444– 456.
Medina, M. A. and Schwille, P. (2002). Fluorescence correlation spectroscopy for
the detection and study of single molecules in biology. BioEssays 24, 758– 764.
Miyawaki, A. (2003). Fluorescence imaging of physiological activity in complex
systems using GFP-based probes. Curr. Opin. Neurobiol. 13, 591– 596.
Murphy, D. B. (2001). Fundamentals of Light Microscopy and Electronic Imaging.
Wiley-Liss, New York.
Oparka, K. J. and Read, N. D. (1994). The use of fluorescent probes for studies on
living plant cells. In Plant Cell Biology. A Practical Approach (N. Harris and K. J.
Oparka, eds), pp. 27 – 50. IRL Press, Oxford.

Live-cell Imaging of
Parton, R. M. and Read, N. D. (1999). Calcium and pH imaging in living cells. In

Filamentous Fungi
Light Microscopy in Biology (A. J. Lacey, ed.), pp. 211 – 264. Oxford University
Press, Oxford, UK.
Parton, R. M., Fischer, S., Malhó, R., Papasouliotis, O., Jelitto, T. C., Leonard, T. and
Read, N. D. (1997). Pronounced cytoplasmic pH gradients are not required for
tip growth in plant and fungal cells. J. Cell Sci. 110, 1187–1198.
Pawley, J. B. (1995). Handbook of Biological Confocal Microscopy. Plenum Press,
New York.
Periasamy, A. and Day, R. N. (1999). Visualizing protein interactions in living cells
using digitized GFP imaging and FRET microscopy. Meth. Cell Biol. 58, 293– 313.
Read, N. D. and Hickey, P. J. (2001). The vesicle trafficking network and tip growth
in fungal hyphae. In Cell Biology of Plant and Fungal Tip Growth (A. Geitmann,
M. Cresti and I. B. Heath, eds), pp. 137– 148. IOS Press, Amsterdam.
Read, N. D. and Kalkman, E. R. (2003). Does endocytosis occur in fungal hyphae?
Fungal Genet. Biol. 39, 199– 203.
Read, N. D., Allan, W. T. G., Knight, H., Knight, M. R., Knight, R., Malhó, R., Russell,
A., Shacklock, P. S. and Trewavas, A. J. (1992). Imaging and measurement of
cytosolic free calcium in plant and fungal cells. J. Microsc. 166, 57–86.
Roca, M. G., Davide, L. C., Mendes-Costa, M. C. and Wheals, A. (2003). Conidial
anastomosis tubes in Colletotrichum. Fungal Genet. Biol. 40, 138– 145.
Roncero, C. and Duran, A. (1985). Effect of calcofluor white and congo red on
fungal wall morphogenesis: in vivo activation of chitin polymerization.
J. Bacteriol. 170, 1950– 1954.
Sheppard, C. J. R. and Shotton, D. M. (1997). Confocal Laser Scanning Microscopy.
IOS Scientific Publishers, Oxford.
Silverman-Gavrila, L. B. and Lew, R. R. (2000). Calcium and tip growth in
Neurospora crassa. Protoplasma 213, 203– 214.
Silverman-Gavrila, L. B. and Lew, R. R. (2001). Regulation of the tip-high [Ca2þ]
gradient in growing hyphae of the fungus Neurospora crassa. Eur. J. Cell Biol. 80,
379–390.
Silverman-Gavrila, L. B. and Lew, R. R. (2003). Calcium gradient dependence of
Neurospora crassa hyphal growth. Microbiology 149, 2475– 2485.
Steinberg, G., Schliwa, M., Lehmler, C., Bölker, M., Kahmann, R. and McIntosh, J. R.
(1998). Kinesin from the plant pathogenic fungus Ustilago maydis is involved
in vacuole formation and cytoplasmic migration. J. Cell Sci. 111, 2235– 2246.
Swedlow, J. R. (2003). Quantitative fluorescence microscopy and image deconvo-
lution. Meth. Cell Biol. 72, 346– 367.
Swift, S. R., Hart, C. A., Bartlett, D. W. and Read, N. D. (2001). Interactions between
azoxystrobin and Puccinia recondita, Erysiphe graminis, and Botrytis cinerea on the
microscale. Scanning 23, 153– 154.

86
Torralba, S. and Heath, I. B. (2002). Analysis of three separate probes suggests the
absence of endocytosis in Neurospora crassa hyphae. Fungal Genet. Biol. 37,
221–232.
Vida, T. A. and Emr, S. D. (1995). A new vital stain for visualizing vacuolar
membrane dynamics and endocytosis in yeast. J. Cell Biol. 128, 779– 792.
Zhang, J., Campbell, R. E., Ting, A. Y. and Tsien, R. Y. (2002). Creating fluorescent
probes for cell biology. Nat. Rev. Mol. Cell Biol. 3, 906– 918.

Live-cell Imaging of
Filamentous Fungi

87