CELL I MAGI NG

THE FI ELD OF CELL BI OLOGY ORI GI NATED WI TH THE DEMONSTRATI ON

OF THE ~CELL THEORY OF LIVING THINGS. THIS DEMONSTRATION
AND THE CONTI NUED DEVELOPMENT OF THE FI ELD WAS SI GNI FI CANTLY
DEPENDENT UPON THE CREATI ON OF TECHNI QUES THAT ALLOWED
VI SUALI ZATI ON OF THE CELLS BEI NG STUDI ED.
THREE FACTORS ARE I MPORTANT FOR THE VI SI BI LI TY OF SMALL
OBJECTS:
1) CONTRAST - THE DI FFERENCE I N APPEARANCE BETWEEN NEI GHBORI NG
PARTS OF THE OBJECT.
2) MAGNI FI CATI ON - TO WHAT DEGREE THE APPARENT SI ZE OF THE
OBJECT CAN BE EXPANDED
3) RESOLUTI ON - HOW WELL CLOSELY SPACED OBJECTS CAN BE
SEEN AS SEPARATE.
FOR OBJECTS OF DI FFERENT SI ZES, DI FFERENT TECHNI QUES ARE REQUI RED. MANY ASPECTS
OF CELL BI OLOGY CAN BE STUDI ED USI NG VARI ATI ONS ON VI SI BLE LI GHT MI CROSCOPY
Microscopy
The basics
1. A source of illumination
- visible light (400 700 nm)
- uv light (200 300 nm)
- electrons (0.004 nm)
2. A system of lenses to focus illumination on the specimen
- glass lenses
- electromagnetic lenses
3. A detector
- eye
- photographic film
- electronic detector (e.g., video camera)
THE STANDARD LI GHT MI CROSCOPE
1 THE CONDENSER LENS GATHERS
DI FFUSE LI GHT RAYS TO I LLUMI NATE
THE SPECI MEN
2 THE SPECI MEN MAY ABSORB, REFRACT
i.e. ALTER OR HAVE NO EFFECT ON THE
I NCI DENT LI GHT
3 THE OBJECTI VE LENS COLLECTS
LI GHT THAT HAS AND HAS NOT BEEN
ALTERED BY THE SPECI MEN. THE
ALTERED RAYS ARE FOCUSED AS AN
ENLARGED I MAGE I N THE TUBE.
4 THE I MAGE FORMED BY THE OBJECTI VE LENS
I S USED AS AN OBJECT BY THE OCULAR LENS
WHI CH CREATES A FURTHER MAGNI FI ED
I MAGE.. THI S I MAGE I S THEN FOCUSED BY THE
LENS OF THE EYE ON THE OBSERVER'S RETI NA
LI GHT AND I MAGE FORMATI ON
AS LI GHT PASSES THROUGH AN OBJECT THE WAVES CAN BE ALTERED
I N DI FFERENT WAYS AND THUS TRAVEL DI FFERENT PATH LENGTHS TO
THE POI NT WHERE THEY ARE RECOMBI NED AS AN I MAGE. HERE, THEY
CAN BE I N PHASE, ADD AND PRODUCE A BRI GHT SPOT OR BE OUT OF
PHASE AND PRODUCE A DI M OR BLACK SPOT. THE I MAGE THEN I S
CONSTRUCTED FROM THIS ~DIGITAL PATTERN.
BECAUSE OF THE WAVE NATURE OF LI GHT,
I NTERFERENCE PATTERNS ARE PRODUCED AT THE
EDGES OF OB1ECTS.. THUS AT THE ~MICRO SCALE
EDGES ARE NOT SHARP AND POI NTS ARE
REPRESENTED AS ~BLURRY DISKS..
AT SI ZES COMPARABLE TO THE WAVELENGTHS OF LI GHT
I NDI VI DUAL POI NTS WI LL APPEAR AS EXPANDED DI SKS.THUS
WHEN THOSE POI NTS ARE CLOSE TOGETHER THAN THE EXPANSI ON
OF THE DISK, THOSE POINT CANNOT BE ~RESOLVED AND WILL
APPEAR AD A SI NGLE OBJECT e.g. SQUARE BELOW.
MAGNI FI CATI ON CANNOT OVERCOME THE RESOLUTI ON
LI MI T. BELOW THE OCULAR LENS I S CHANGED LEADI NG
TO AN I NCREASE I N MAGNI FI CATI ON ALTHOUGH I T
BECOMES POSSI BLE TO SEE AND COUNT THE NUMBER OF
~CHROMOSOMES NO ADDITIONAL DETAILS OF
CHROMOSOME STRUCTURE BECOME VI SI BLE.
RESOLUTI ON
THE RESOLVI NG POWER OF A MI CROSCOPE I S A MEASURE OF I TS ABI LI TY TO SEE TWO
NEI GHBORI NG POI NTS I N THE VI SUAL FI ELD AS DI STI NCT ENTI TI ES. THI S QUALI TY I S
DEPENDENT UPON THE OBJECTI VE LENS OF THE SCOPE. PHYSI CALLY THE RESOLVI NG POWER
I S LI MI TED BY THE WAVELENGTH OF THE I NCI DENT LI GHT AND HAS BEEN FOUND TO CHANGE
ACCORDI NG TO THE FOLLOWI NG EQUATI ON.
0.61
d = nsin
Where:
d is the minimum distance two points must
be separated to be resolved
is the wavelength of the light ( 527nm is
used for ~white light)
n is the refractive index of the medium
between the objective lens and the
specimen.
is equal to the angle of the cone of the
light entering the objective lens
The denominator in the above equation nsinis called the NUMERI CAL APERTURE, which is a measure
of the quality i.e. the light gathering properties of the objective lens.
For a lens that acts in air (n=1), and designed for the max light gathering ability (1), the NA is equal to 1 the
best possible. For an oil lens, where n can be greater than 1 the max NA is about 1.5. Such high NA lenses are
designed to have short focal length I .e. the distance of lens to specimen or virtual image.
When we substitute the shortest visible wavelength and the highest NA attainable we find that the Limit of
Resolution of the light microscope is 200nm sufficient to resolve organelles such as nuclei or mitochondria
Bright-Field Microscopy
THE MOST COMMON FORM OF MI CROSCOPY I S BRI GHT-FI ELD. HERE, THE I MAGE I S
OBTAI NED BY THE SI MPLE TRANSMI SSI ON OF THE LI GHT THROUGH THE SPECI MEN.
A LI MI TATI ON I S THAT LI VI NG TI SSUE OR CELLS SHOW LI TTLE VARI ATI ON I N OPTI CAL
DENSI TY AND THUS LI TTLE CONTRAST WI TH WHI CH TO BUI LD AN I MAGE. TO OVERCOME
THI S CELLS/TI SSUES ARE FI XED, SECTI ONED AND THEN STAI NED. SI NCE DI FFERENT AREAS
OF THE CELL/TI SSUE BI ND DI FFERENT DYES OR AMOUNTS OF DYES CONTRAST CAN BE
ENHANCED. HOWEVER, CARE MUST BE TAKEN TO I NSURE THAT THE PROCESS OF
PREPARATI ON DOES NOT DI STORT THE ORI GI NAL ANATOMY OF THE SPECI MEN.
A HI GH MAG VI EW OF A CELL STAI NED WI TH
HEMATOXALI N, A NUCLEI C ACI D BI NDI NG DYE.
THI S PROCESS MAKES I NDI VI DUAL
CHROMOSOMES CLEARLY VI SI BLE
A LOW MAG VI EW OF A FI XED, SECTI ONED AND STAI NED
TI SSUE, AS MI GHT BE USED I N HI STOPATHOLOGY. THE
STAI N, HERE, I S THE CONVENTI ONAL HEMATOXALI N AND
EOSI N (H&E). NUCLEI ARE BLUI SH AND CYTOPLASMI C AND
MANY EXTRACELLULAR COMPONENTS RED..
I NTERFERENCE MI CROSCOPY
I N THE FI GURES BELOW NOTE THE DI FFERENCE I N THE LI GHT PATHS. FOR THE PHASE SCOPE A DI APHRAGM
CONVERTS THE SOURCE TO A RI NG. AS THI S RI NG PASSES THROUGH THE SPECI MEN MOST LI GHT I S UNALTERED
(YELLOW) BUT SOME I S REFRACTED BY VARI ATI ONS I N THE SPECI MEN (BLUE) WHEN THEY ARE RECOMBI NED
AT THE IMAGE PLANE THEY WILL BE SIGNIFICANTLY ~OUT OF PHASE SETTING UP LIGHT AND DARK AREAS,
WHICH REFLECT DIFFERENT DENSITIES AND ~EDGES OF THE SPECIMEN. SUCH A METHOD CAN GIVE AN IMAGE
OF A LIVE, UNSTAINED CELL. THUS ~PHASE-CONTRAST IS COMMONLY USED TO OBSERVE CULTURED CELLS.
SI MI LAR I N PRI NCI PLE, BUT MORE COMPLEX I N THEORY AND PRACTI CE ARE THE TECHNI QUE OF DI FFERENTI AL
I NTERFERENCE CONTRAST (DI C OR NOMARSKI OPTI CS). I N SOME CASES THE TECHNI QUE CAN BE FURTHER
ENHANCED BY USI NG A DI GI TAL VI DEO CCD CAMERA TO CAPTURE THE I MAGE. SI NCE MULTI PLE DI GI TAL
FRAMES CAN BE RECORDED, COMPUTER BASED TECHNI QUES CAN BE EMPLOYED TO PROCESS THE I MAGE.
WHI TE LI GHT LEVEL I MAGI NG
BRI GHT-FI ELD
PHASE-CONTRAST
DI FFERENTI AL I NTERFERENCE CONTRAST
DARK-FI ELD
VI DEO ENHANCED DI C
THIS IS A ~FRAME FROM
A PREPARATI ON OF ACTI N
FI LAMENTS. VI DEO
ENHANCEMENT ALLOWS
~VISUALIZATION OF
OBJECTS TECHNI CALLY
BELOW THE ~LIMIT OF
RESOLUTION
FLUORESCENCE MI CROSCOPY
SOME CHEMICAL COMPOUNDS CAN ABSORB ~INVISIBLE ULTRAVIOLET RADIATION AND THEN
RELEASE SOME OF THI S ENERGY I N THE LONGER VI SI BLE WAVELENGTH RANGE. THI S
PHENOMENON IS CALLED FLUORESCENCE. IF A ~FLUOROCHROME IS INCORPORATED INTO
A CELL AND EXPOSED TO UV RADI ATI ON THE STAI NED OBJECT WI LL APPEAR COLORED AGAI NST
A BLACK BACKGROUND i.e. AT HI GH CONTRAST.
FLUORESCENCE MI CROSCOPES TAKE ADVANTAGE OF THI S PHYSI CS BY USI NG A HI GH I NTENSI TY
UV LI GHT SOURCE AND A DI CHROI C MI RROR, WHI CH REFLECTS UV (to the specimen) WHI LE
ALLOWI NG THE EMI TTED LI GHT TO PASS AND BE COLLECTED TO FORM AN I MAGE OF THE
OBJECT.
FLUORESCENCE I MAGI NG
HERE, A FLUOROCHROME HAS BEEN TAKEN UP I NTO A LI VI NG CELL. HOWEVER, I T ONLY
FUNCTI ONS I N THE PRESENCE OF Ca I ONS. THUS PHYSI OLOGI CAL STUDI ES BECOME
POSSI BLE i.e. A TREATMENT THAT I NDUCES THE UPTAKE OF Ca CAN BE FOLLOWED I N BOTH
TI ME AND SPACE.
I MMUNOFLUORESCENCE
THE MOST COMMON FLUORESCENCE TECHNI QUE I S PROBABLY I MMUNOFLUORESCENCE.
HERE, AN ANTI BODY I S PRODUCED AGAI NST A CELLULAR COMPONENT OF I NTEREST. THE CELL
OR TI SSUE I S THEN FI XED AND PERMEABLI ZED. THE PRI MARY ANTI BODY I S THEN APPLI ED
TO ATTACH TO THE CELL COMPONENT OF I NTEREST. THI S I S THEN VI SUALI ZED BY USE OF A
SECONDARY ANTI BODY COUPLED TO A FLUOROCHROME.
A MODERN VARI ATI ON EMPLOYS GFP (GREEN FLUORESCENT PROTEI N). THI S SMALL PROTEI N
CAN BE COUPLED TO A CELLULAR COMPONENT OF I NTEREST BY RECOMBI NANT DNA
TECHNI QUES. THE RESULTI NG CHI MERI C GENE OR PROTEI N CAN BE I NCORPORATED I NTO A
CELL WHERE I T WI LL (HOPEFULLY) BEHAVE AS THE TARGET PROTEI N WOULD I N A NORMAL
CELL.
I MMUNOFUORESCENT I MAGES
I N THI S CELL SPI NDLE MI CROTUBULES
ARE STAI NED WI TH A GREEN
FLUORESCENT ANTI BODY, THE
CENTROMERES WI TH A RED
FLUORESCENT ANTI BODY AND THE
CHROMOSOMES WI TH A BLUE DNA
BI NDI NG DYE
I N THI S TI SSUE SECTI ON A SUB-MEMBRANE
CELL CELL CONTACT PROTEI N I S LABELED
WI TH A FI TC CONJUGATED ANTI BODY
I N THI S PANEL A GFP CONJUGATED PROTEI N I S
USED I N A STUDY OF ER/GOLGI I NTERACTI ON
AN EM AND AN ANTI -TUBULI N I MMUNO
I MAGE OF THE SAME REGI ON OF A
CULTURED CELL
CONFOCAL SCANNI NG MI CROSCOPY
MANY OPTI CAL I MAGES SUFFER FROM THE FACT THAT THE OBJECT UNDER STUDY I S THREE
DI MENSI ONAL AND LI GHT FROM OUT OF FOCUS REGI ONS BLURS THE I MAGE. THI S CAN BE
COMPENSATED FOR BY ~CONFOCAL SCANNING, HERE, ILLUSTRATED FOR FLUORESCENCE.
A HI GH I NTENSI TY LI GHT SOURCE, i.e. A LASER I S USED. THE I NCI DENT LI GHT I S LI MI TED TO A TI NY SPOT BY A PI NHOLE. AND FOCUSED
BY THE LENS ON ONE POINT IN ONE ~FOCAL PLANE OF THE SPECIMEN. THE BEAM IS THEN MOVED TO ILLUMINATE A NEIGHBORING
POINT i.e. IT IS ~SCANNED ACROSS THE SPECIMEN. THE LIGHT EMITTED BY THE SPECIMEN IS BROUGHT TO A FOCAL POINT WITHIN THE
SCOPE PRECISELY AT A SECOND PINHOLE I.e. THE PLANE OF THE PINHOLE IS ~CONFOCAL WITH THE PLANE OF THE ~OPTICAL SECTION
OF THE SPECI MEN. THUS ONLY LI GHT FROM THE STUDI ED PLANE AND NOT THAT FROM ABOVE OR BELOW REACHES THE DETECTOR
AND CONTRI BUTES TO THE I MAGE.
CONFOCAL SCANNI NG I MAGES
BY SELECTI NG LI GHT FROM ONLY ONE FOCAL PLANE THE I MAGE CLARI TY I S SI GNI FI CANTLY
ENHANCED.
Figure 1.31 Confocal Micrograph of
Human Cells
COMPUTER ENHANCEMENT
HERE, MULTI PLE CONFOCAL FLUORESCENT I MAGES OF THE SAME CELL ARE COMBI NED I NTO
A SI NGLE I MAGE BY A COMPUTER PROGRAM. SUCH RECONSTRUCTI ONS CAN ALSO BE DONE FOR
DATA FOR A SI NGLE COMPONENT BUT OVER MULTI PLE FOCAL PLANES, THUS GENERATI NG A 3D
I MAGE. I NDEED BY USI NG VI DEO TECHNI QUES ON LI VI NG CELLS A 4D I MAGE i.e. CELL
STRUCTURE CHANGI NG OVER TI ME CAN BE CONSTRUCTED.