COURSE OF BIOPHYSICS .

B I O T E C H N O L O G Y. U A E M É X
• Also called fluorimetry, it is a type of spectroscopy based on
the fluorescent emission of a sample. This involves the use of a
beam of light, commonly ultraviolet light, that excites electrons
in certain molecules or atoms and causes the emission of light,
typical but not necessarily visible light.
His great sensitivity, the sensitivity of fluorescence-based methods is
10 to 10,000 times higher, which means that nanograms can be
analyzed on a picogram by picogram from various analytes with very
good results.

The high level of evolution achieved by both the required instruments,

as well as fluorophores designed for specific applications.

Its specificity, which allows specific molecules to be identified in

complex matrices.
•SPECTROFLUOROMETER
 The molecules have
different states called
energy levels refers
mainly to vibrational
and electronic states.

The sample is first excited by

the absorption of a light
photon, from its basal Collisions with other molecules cause the excited molecule to
electronic state to one of lose vibrational energy until it reaches the lowest vibrational
the different vibrational state of the excited electronic state.
states of the excited
electronic state.

By analysing the different frequencies of

The molecule then
descends to one of the
light emitted by fluorescence
different vibration levels of spectrometry, together with their relative
the basal electronic state, intensities, the structure of the different
emitting a photon in the levels of vibration can be determined.
process.
Una molécula o parte
de una molécula que
emite fluorescencia
después de ser
excitada con luz.
Dichas moléculas son
generalmente
orgánicas
poliaromáticas o
Los fluoróforos heterociclos.
se pueden
dividir en dos
clases generales:
extrínsecos e
intrínsecos.
• When the fluorophore absorbs
light one of its electrons passes
into an excited (higher energy)
state that is unstable and when
it returns to its basal state, the
excess energy is released in
the form of light but of a
longer wavelength (lower
energy) than that of excitation.
This process is represented by
the Perrin-Jablonski diagram.
 • In fluorescence spectroscopy
excitation and emission spectra are
recorded. The excitation spectrum
corresponds to the absorbance
spectrum. Both spectra are a
representation of the fluorescence
intensity in arbitrary units as a
function of the wavelength in nm. An
excitation wavelength is fixed for
recording the emission spectrum and
an emission wavelength for
excitation.
DNA SEQUENCING
SEQUENCING SYSTEM SCHEME USING FLUOROPHORES COUPLED TO MODIFIED NUCLEOTIDES
MEMBRANE FUSION
S C H E M AT I C R E P R E S E N TAT I O N O F T H E F U S I O N O F F L U O R E S C E N T LY M A R K E D M E M B R A N E S . D I S
THE DONOR MOLECULE AND A IS THE ACCEPTOR MOLECULE. THE DECREASE IN THE
E F F I C I E N C Y O F F R ET I S R E G I S T E R E D B Y A N I N C R E A S E I N T H E I N T E N S I T Y O F T H E
F L U O R E S C E N C E O F T H E D O N O R A N D T H E R E F O R E T H E D E C R E A S E I N T H AT O F T H E A C C E P T O R .
FLUORESCENCE OF PROTEINS.
PRINCIPLE OF FLUORIGENIC RESPONSE TO PROTEASE CUTTING. FLUOROPHORE
(F) AND DAMPER (Q).
REAL-TIME PCR.
R E A L - T I M E P C R S C H E M AT I C R E P R E S E N TAT I O N W I T H TA Q M A N O L I G O S . T H E G R E E N C I R C L E
REPRESENTS THE FLUOROPHORE, WHILE THE RED CIRCLE REPRESENTS THE DAMPER..