CELL
Reference:
Biol. Bull. 187: 231-232.
(October,
DIVISION,
CELL
MOTILITY,
AND
231
DEVELOPMENT
1994)
High Resolution Multimode Digital Imaging System for Mitosis Studies In Viva and In Vitro
E. D. Salmon (Biology, Univ. of N. Carolina, Chapel Hill, NC), T. Inouk, A. Desai, and A. W. Murray
Many questions about the mechanisms of mitotic spindle assembly, chromosome movement, and chromosome segregation
can be answered by quantitative measurements of digital images
obtained from several optical modes. For example, epifluorescent
detection of X-rhodamine-labeled tubulin reveals spindle microtubule organization, whereas DAPI or other DNA-binding
dyes stain the chromosomes in live preparations where these
structures are often invisible to phase or DIC methods (l-4).
Photoactivated fluorophores bound to tubulin can be used to
measure microtubule assembly dynamics in real time (1,2), and
other fluorescent probes can be used to track the dynamics of
membranes, other organelles, or molecular complexes within
the spindle (5). In transparent specimens, the movements of
chromosomes and centrosomes can be recorded with transmitted
light if phase contrast or DIC optics are employed (6) while
polarization methods can reveal spindle fiber microtubule dynamics and provide a quantitative measure of microtubule assembly and orientation (6). In addition to real-time recording,
a digital fluorescence microscope system can also provide 3-D
structural detail from stacks of optical sections when the specimens are stained with specific molecular probes.
The high resolution, multimodal digital microscope system
that we have constructed for our mitosis studies is diagrammed
in Figure 1. Optical components of the Nikon FXA microscope
stand were chosen to provide diffraction-limited resolution in
transmitted and fluorescence modes for images projected onto
a cooled charge coupled device (CCD) camera (Fig. 2C).
Cooled CCD cameras have several advantages for digital imaging over unintensified and intensified video detectors, including dynamic range, linearity, low noise, and little geometrical
distortion. The Hamamatsu C4880 was chosen because it has
two readout modes: a fast scan mode (up to 7 frames/s) which
is useful for focusing, and a slow scan mode ( 12 bit/pixel,
500,000 bytes/s readout rate), which gives the most useful dynamic range (4000 grey levels above a noise floor of about
50 f 10). The MetaMorph digital imaging system is programmed to control image acquisition from the CCD camera.
We have found that the central area (300 X 300 pixels) of the
1000 X 1000 pixel detector provides sufficient resolution and
field of view for our mitosis images while reducing the time
required to process the digital images and the amount of digital
storage required (180 KBytes/image).
An important criterion in our mitosis studies is that all the
epi- and trans-illumination images be aligned and in focus at
the same position on the CCD detector. This was accomplished
with a single Chroma filter cube containing a multiple bandpass
dichromatic mirror and emission filters designed for the DAPI
(blue), fluorescein (green), and X-rhodamine (red) emission
wavelengths. Excitation intensity and wavelength are selected
by a MetalTek stepper motor controlled dual filter wheel, where
one g-position wheel holds a series of neutral density filters, and
the other holds narrow bandpass filters for the different excitation
wavelengths. Other filters and iris diaphragms in the epi- and
trans-illumination paths are used to provide further manual
control of illumination intensity and field of illumination (Fig.
1). Focus position along the z-axis is controlled by a Ludl stepper
motor attached to the Nikon FXA fine focus.
The MetaMorph digital imaging system has programs and
journal scripts that control for either time-lapse or z-axis stepping,
CCD
M
A
MBPDEF
F
DFW
S-VHS
I
wo
1,
ST
I
00
,+,
El
C
WC
P
aml
NIKON
FXA
- f3 -I
4
LIGHT
MICROSCOPE
METAMORPH
DIGITAL
IMAGING
SYSTEM
Figure 1. Component
parts are Ll, 100 W quartz halogen lamp; Sl,
Uniblitr shutter (#225L2AlZS23398,
Vincent Associates, Rochester, NY);
FBI, FB2, manualfilter
changers for Nikon FXA stand: I,, Iz. Is. 1,jield
and condenser iris diaphragms:
P, AP, high transmission
Nikon Polaroid
polarizer
and removable
analyzer;
WC, WO, DIC Wollaston prisms; C,
Nikon NA = 1.4 CON A, Achr-Apl
condenser; ST, rotatable stage with
focus position controlled
by z-axis stepper motor (Mac2000.
Ludl Electronic Products, LTD., Hawthorne,
NY); OB, ZOX/NA = .75 or 6OX/NA
= 1.4 Nikon objectives; MBPDEF,
filter block with multiple bandpass
dichromatic
mirror and emission jilter (#83101 and #83100, Chroma
Technology
Corp., Brattleboro,
VT,); L2, 100 W HBO Hg lamp; F, KG4
heat cut jilter;
S2, DFW,
shutter and dual d-position
jilter
wheel
(MetalTek,
Raleigh, NC), one wheel containing
neutral density filters
(#FNQOI
1, Meiles Griot, Irvine, CA), and the other a series of narrow
bandpass excitation filters (#83360, #83490, #83570, Chroma Technology
Corp.); M, optivar magnification
changer, M-2X;
OC, oculars; CCD.
cooled CCD camera (#C4880, Hamamatsu
Photonics, Bridgewater,
NJ);
DD. 1024 X 768 pixel, 20 inch digital graphics display monitor (#2082,
Viewsonic);
VD, RGB video display monitor
(#PVM1271Q~
Sony);
MetaMorph
digital imaging
system (Universal
Imaging
Corp., West
Chester, PA) using a 66 MHz, 486 processor, EISA bus, 64 MByte RAM
memory,
Imaging
Technology AFG digital and video image processing
card; Hamamatsu
C4880 CCD controller
card; Matrox
MGA
Uhima
graphics display card, graphics display to S-VHS converter card (Hyperconverter,
PC Video Conversion
Corp., San Jose, CA), 1.4 MByte
floppy drive, 580 MByte hard drive, Pinnacle Micro 650 MByte optical
drive, Ethernet
card, parallel port cards for controlling
shutter SI and
driving laser printer:
8 serial port card for controlling
MetalTek
filter
wheel, Ludl z-axis stepper, CCD camera, and OMDR.
REPORTSFROM THE MBL GENERAL SClENTIFlCMEETINGS
232
color overlaysof different imagestacks(e.g., DAPI and rhodamine channels),and movie presentation,either on the high resolution graphicsscreenor by conversionof the digital images
to video througha VGA to S-VHS video converter. Largestacks
of imagescanalsobeconvertedto video for storageon an optical
memory video disk recorder(PanasonicTQ2028 or TQ3038F).
Usingthis systemwe wereableto obtain novel highresolution
real-time imagesof yeast nuclear motion in the cell division
cycle (Fig. 2A, B) and to finally visualizein real time anaphase
spindledynamicsand chromosomesegregationin an in vitro
system(3, 4) reconstitutedfrom spermnuclei and extractsprepared from Xenopuseggs(Fig. 2C, D, E). 3-D imagesderived
from a stackof optical sectionshave alsoproved very usefulfor
determiningthe behavior of all of the chromosomesand their
kinetochoreregionswithin the spindle(Fig. 2F).
WegratefullyacknowledgeMBL for providing the H. L. Rand
Fellowshipto E.D.S., MBL and Nikon for providing the Nikon
Fellowshipto A. W. M. and for their generousloan of instruFigure 2. Views of a living, dividing yeast (Saccharomyces
cerevisiae) ments,and Shinya Inoue and the Programin Architectural Dyproduced by (A) green fluorescent protein (7) bound to nuclear htstones
namicsfor support and spacefor our Xenopusstudiesat the
and (B) DIG* (C) Image in DIC of the 0.24 nm spacing between row
MBL. The instrument development and Xenopus studies
of frustrual pores of the diatom Amphipleura
illumrnated wrth green
are supported by NIH GM24364 to E.D.S. and GM 43987
ltght*. Images of a spmdle undergoing anaphase in Xenopus
cytoplasmrc
to A.W.M.
egg extracts: (D) DAPI stained chromosomes, (E) Rhodamme-tub&n
labeled sprndle und aster mrcrotubules. and (F) phase contrast (Ntkon
ZOX/NA = 0 75 Fluar Phase 3 ObJective).Stereo parr Image.7(F) ofDAPI
stamed chromosomes generated from a stack of 0 5 nm optrcal secttons
through a Xenopussprndle fixed rn the extracts m mtd-anaphase*
(*, Ntkon 6OX/NA = I 4 Plan Apo DIG obJectwe and NA = 14 condenser
rllumrnatron for DIC)
shuttersin the light paths,filter wheelpositions,imagesizeand
acquisitionfrom theCCD camera,andimagestorageinto stacks
identified by their mode of acquisition(DAPI, Xrhod, phase,
DIC, etc.). Imagestacksare initially storedwithin the 64 MBytes
of RAM memory,then archivedon the harddrive or the Pinnacle
optical disk drive. MetaMorph also provides comprehensive
functionsfor quantitative analysisof intensityand motion,multi-
Reference:
Biol. Bull.
Through-Focal
187: 232-233.
Literature
Cited
1. Sawin, K. E., and T. J. Mitchison.
1992.
J Cell Biol. 112: 941954.
2. Mitchison,
T. J., and E. D. Salmon. 1992.
J. Cell Btol. 119: 569582.
3. Shamu, C. E., and A. W. Murray.
1992.
J Cell Btol. 117: 921934.
Cell 73: 1393-1402.
4. Hollaway,
S. L., and A. W. Murray.
1993.
5. Waterman-Storer,
C. M., J. W. Sanger,
and J. M. Sanger.
1993. Cell Motil. Cytoskel. 26: 19-39.
CellMotil. Cy6. Cassimeris,
L., S. Inoue, and E. D. Salmon. 1988.
toskel 10: I85- 196.
7. Chalfie, M., Y. Tu, G. Euskirchen,
W. W. Ward, and D. C. Prasher.
1994.
Sctence 263: 802-804.
(October, 1994)
and Time-Lapse
Stereoscopic Imaging
in DIC and Polarization
of Dividing
Microscopy
Cells and Developing
Embryos
Shinya Ino& (Marine Biological Laboratory) and Theodore D. Inoui
Recentdevelopmentsin confocal microscopy,and methods
for computationaldeconvolutionof serialoptical sections,now
permit three-dimensional(3-D) imaging of unfixed cells and
developingembryos,generallyin the fluorescence
contrastmode
(1). 3-D reconstruction is restricted to fluorescencecontrast,
partly because
mostconfocalmicroscopes
do not permit confocal
imagingin other contrast modes,and partly becausethe 3-D
point-spreadfunction for fluorescenceis relatively simple.
Although fluorescencemicroscopy is extremely effective at
revealingspecificchemicalspeciesand selectedorganelles,DIC
and polarizationmicroscopycan providecomplementaryspatial
and fine-structural information, and at much greaterspeeds.
However,the point-spreadfunction in theselatter modesof microscopyis extremely complex (2), and it is not obviousthat 3D imagescan be computationallyreconstructedin thesemodes
from serialoptical sections.
Nevertheless,
we reportedearlierthat very thin opticalsections
can be obtained in DIC, polarization, and phasecontrastmicroscopyin the absenceof confocal imagingby usingwell-correctedoptics of high numericalaperature(NA) and video and