OPTICAL BIO IMAGING

PRESENTED BY
GEORGE P MATHEW
2
ND
SEM MTECH
ISP,CUSAT
OPTICAL BIO IMAGING- BIO IMAGING USING OPTICAL
METHODS.
IT COVERS A BROAD RANGE OF LENGTH SCALE FROM
SUBMICRON SIZE VIRUSES AND BACTERIA TO
MACROSCOPIC SIZED LIVE BIOLOGICAL SPECIES.
OPTICAL BIOIMAGING UTILIZES AN OPTICAL CONTRAST
SUCH AS A DIFFERENCE IN LIGHT TRANSMISSSION
,REFLECTION AND FLUORESCENCE BETWEEN THE REGION
TO BE IMAGED AND THE SURROUNDING REGION.
LIMITATIONS OF CURRENT TECHNIQUES
THESE ARE LARGELY FOCUSED ON STRUCTURAL AND ANATOMICAL
IMAGING AT THE TISSUE OR ORGAN LEVELS.ONLY INFORMATION AT
THE MOLECULAR AND CELLULAR LEVELS LEAD TO THE DETECTION
OF THE EARLY STAGES OF THE FORMATION OF A DISEASE OR
CANCER.
HARMFUL EFFECTS OF RADIATIONS IN THE CASE OF X-RAY
IMAGING & CAT SCAN.

UN SUITABILITY OF X-RAY IMAGING FOR YOUNG PATIENTS.
INABILITY OF MRI TO PROVIDE SPECIFIC CHEMICAL INFORMATION
AND ANY DYNAMIC IFORMATION.
INABILITY OF ULTRASOUND TO PROVIDE RESOLUTION SMALLER
THAN MILLIMETERS.
ADVANTAGES OF OPTICAL IMAGING
NOT BEING HARMFUL.
IMAGING FROM SIZE SCALE OF 100NM TO MACROSCOPIC
OBJECTS.
MULTIDIMENSIONAL IMAGING.
INFORMATION ON CELLULAR PROCES AND TISSUE CHEMISTRY.
ABILITY TO COMBINE OPTICAL IMAGING WITH OTHER
TECHNIQUES.
MORE SENSITIVITY AND SELECTIVITY.
OPTICAL METHODS OF IMAGING
PROPAGATION OF LIGHT THROUGH TURBID MEDIUM
TRANSILLUMINATION METHODS
SPATIAL FILTERING USING CONFOCAL APERTURE (PIN HOLE)
POLARIZATION GRATING USNG POLARIZER
TIME GATING USING OPTICAL GATE
FREQUENCY DOMAIN METHODS USING INTENSITY MODULATED
BEAM
REFLECTION IMAGING
USING BACK SCATTERED LIGHT
FLUORESCENCE MICROSCOPY

TRANSMISSION MICROSCOPY
OPTICAL MICROSCOPE
IT IS A TYPE OF MICROSCOPE WHICH USES VISIBLE LIGHT AND A
SYSTEM OF LENSES TO MAGNIFY IMAGES OF SMALL SAMPLES. THE
IMAGE FROM AN OPTICAL MICROSCOPE CAN BE CAPTURED BY
NORMAL LIGHT-SENSITIVE CAMERAS TO GENERATE A
MICROGRAPH. ORIGINALLY IMAGES WERE CAPTURED BY
PHOTOGRAPHIC FILM BUT MODERN DEVELOPMENTS IN CMOS AND
CHARGE-COUPLED DEVICE (CCD) CAMERAS ALLOW THE CAPTURE
OF DIGITAL IMAGES.
OPTICAL CONFIGURATIONS
1. SIMPLE MICROSCOPE
A SIMPLE MICROSCOPE IS A MICROSCOPE THAT USES A LENS
TO ENLARGE AN OBJECT THROUGH ANGULAR MAGNIFICATION
ALONE, GIVING THE VIEWER AN ERECT ENLARGED VIRTUAL IMAGE.
IT CONSIST OF SINGLE LENS MOUNTED ON A METAL PLATE WITH
SCREWS TO MOVE THE SPECIMEN ACROSS THE FIELD OF VIEW AND
TO FOCUS ITS IMAGE.
COMPOUND MICROSCOPE
IT CONSISTS OF A COMBINATION OF LENSES TO IMPROVE THE
MAGNIFICATION. IT USES A LENS CLOSE TO THE OBJECT BEING
VIEWED TO COLLECT LIGHT (CALLED THE OBJECTIVE LENS) WHICH
FOCUSES A REAL IMAGE OF THE OBJECT INSIDE THE MICROSCOPE .
THAT IMAGE IS THEN MAGNIFIED BY A SECOND LENS OR GROUP OF
LENSES (CALLED THE EYEPIECE) THAT GIVES THE VIEWER AN
ENLARGED INVERTED VIRTUAL IMAGE OF THE OBJECT
SCHEMATIC DIAGRAM OF TRANSMISSION MICROSCOPE
ILLUMINATION TECHNIQUES
A PROPER ILLUMINATION OF THE SPECIMEN OBSERVED UNDER THE
MICROSCOPE IS CRITICAL FOR ACHIEVING A HIGH QUALITY IMAGE
THROUGH THE EYEPIECE.

KOHLER ILLUMINATION
KHLER ILLUMINATION IS A METHOD OF SPECIMEN ILLUMINATION
USED FOR TRANSMITTED OPTICAL MICROSCOPY. KHLER
ILLUMINATION ACTS TO GENERATE AN EXTREMELY EVEN
ILLUMINATION OF THE SAMPLE AND ENSURES THAT AN IMAGE OF
THE ILLUMINATION SOURCE IS NOT VISIBLE IN THE RESULTING
IMAGE
SCHEMATIC DIAGRAM FOR KOHLER ILLUMINATION
PHASE CONTRAST MICROSCOPY
THIS TECHNIQUE IS BEST FOR LOOKING AT LIVING SPECIMENS, SUCH AS
CULTURED CELLS.CELLS DONT INTRODUCE ANY AMPLITUDE CHANGE IN
THE TRANSMITTED LIGHT. HENCE ARE DIFFICULT TO OBSERVE IN NORMAL
BRIGHT FIELD MICROSCOPY. IT PROVIDES ENHANCEMENT OF CONTRAST. IN
A PHASE-CONTRAST MICROSCOPE, THE LIGHT THAT PASSES THROUGH THE
CENTRAL PART OF THE LIGHT PATH IS RECOMBINED WITH THE LIGHT THAT
TRAVELS AROUND THE PERIPHERY OF THE SPECIMEN. THE INTERFERENCE
PRODUCED BY THESE TWO PATHS PRODUCES IMAGES IN WHICH THE DENSE
STRUCTURES APPEAR DARKER THAN THE BACKGROUND.
OPTICAL SCHEMATICS FOR PHASE CONTRAST MICROSCOPY
DARK FIELD MICROSCOPY
DARK FIELD MICROSCOPY DESCRIBES MICROSCOPY METHODS,
WHICH EXCLUDE THE UNSCATTERED BEAM FROM THE IMAGE. AS A
RESULT, THE FIELD AROUND THE SPECIMEN IS GENERALLY DARK. IT
WORKS BY ILLUMINATING THE SAMPLE WITH LIGHT THAT WILL NOT
BE COLLECTED BY THE OBJECTIVE LENS, AND THUS WILL NOT
FORM PART OF THE IMAGE. THIS PRODUCES THE CLASSIC
APPEARANCE OF A DARK, ALMOST BLACK, BACKGROUND WITH
BRIGHT OBJECTS ON IT.
DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY
IT IS AN OPTICAL MICROSCOPY ILLUMINATION TECHNIQUE USED
TO ENHANCE THE CONTRAST IN UNSTAINED, TRANSPARENT
SAMPLES. DIC USES POLARIZING FILTERS AND PRISMS TO
SEPARATE AND RECOMBINE THE LIGHT PATHS, GIVING A 3-D
APPEARANCE TO THE SPECIMEN. IT IS A TECHNIQUE THAT
CONVERTS SPECIMEN OPTICAL PATH GRADIENTS IN TO AMPLITUDE
DIFFERENCES THAT CAN BE VISUALISED AS IMPROVED CONTRAST
CONCLUSION
THE AREA OF OPTICAL IMAGING IS VERY RICH BOTH IN TERMS OF
THE NUMBER OF MODALITIES AND WITH REGARD TO THE RANGE OF
ITS APPLICATIONS. IT IS ALSO AN AREA OF VERY INTENSE
RESEARCH WORLDWIDE BECAUSE NEW METHODS OF OPTICAL
IMAGING , NEW INSTRUMENTATIONS AND NEW APPLICATIONS ARE
CONSTANTLY EMERGING.