Basic Application of Fluorescence in biology

1. Presented by- Budha Ratna Rav
MP17001
Fluorescence spectroscopy: Application in Biological studies

2. Basic principle of fluorescence spectroscopy

3. Principle of Fluorescence microscopy

4. Gal4 expression induces the UAS2xeGFP expression inside the cell.
Simply set up the cross between the two different genotype populations of Drosophila melanogaster:
Female Male
w//w; +//+; DppGal4//Tb × w//y; UAS2xeGFP//UAS2xeGFP; +//+
W//W; UAS2xeGFP//+; DppGal4//+ (Red eye color female, GFP expressed inside cells, Non- tubby)
W//y; UAS2xeGFP//+; DppGal4//+ (Red eye color male, GFP expressed inside cells, Non- tubby)
W//W; UAS2xeGFP//+; Tb//+ (Red eye color female, No expression of Reporter gene, Tubby)
W//y; UAS2xeGFP//+; Tb//+ (Red eye color female, No expression of reporter gene, Tubby)
Study of Reporter gene- expression
Leg Imaginal disc of drosophila
with expression of GFP
Paper Link: http://www.pnas.org/content/pnas/92/15/7036.full.pdf
Reference: EDWARD YEH*, KERSTIN GUSTAFSON*, AND GABRIELLE L. BOULIANNEt

5. ELAV is the neuron specific protein.
During development of animal
neurulation is happen before
organogenesis.
Primary Antibody specific to ELAV-
protein.
Secondary Antibody specific to Fc-
region of anti-ELAV antibody which
labelled with fluorescent dye.
Immunostaining of Drosophila embryos by anti-ELAV antibody
Lateral view of Drosophila embryo
Ventral view of Drosophila embryo
Antibody staining of whole mount Drosophila embryos
By Dr. Torsten Bossing, Wellcome Trust / CRUK Gurdon Institute, U.K.

6. Labelled the dNTPs(dATP, dTTP,
dCTP, dGTP) with Fluorescent
dye.
Detectors detect the DNA
sequence.
Application of fluorescent in DNA sequencing

7. Study of protein-protein interaction
Produce two fusion proteins with two different
fluorophore.
Emission spectrum of one fluorochrome must overlap with
absorption spectrum of other fluorochrome.
Study of Nucleic Acid annealing:
Two complementary oligonucleotides are labelled with Cy3
and Cy5
Application of FRET

8. Measurement the rate of lateral diffusion of
membrane
This technique composed of three phase-
1) Prebleach phase: Specific proteins are
labelled with fluorescent dye.
2) Photobleaching phase: Labelled proteins are
bleached in small area using laser beam.
3) Postbleach phase: If diffusion occur
fluorescent intensity will observe in
bleached area.
Study of lateral diffusion in Plasma membrane by (FRAP)
Basic Mechanism of FRAP

9. Fluorescent In-situ Hybridization (FISH)
FISH used to detect and localize the presence
or absence of
specific DNA sequences on chromosomes.

10. Fluorescent Activating Cell sorting (FACS)
Study for number of cells expressing
particular protein.
Further help in study of particular cells.