Focal plane of lens

1
object
image
detector
full field microscope, magnification defined by
image distance/object distance
scanning microscope, magnification defined by
display size / scan size
image
Focal plane of lens
In epi-illuminated light microscopy, , all arrows
are double headed.
Focal plane of lens
object
object
2

• Probe formation and signal collection in light
  microscopy (case study using Marshall U. MRC 1024
  CSLM)

laser in, signal from sample out
In conventional microscopy the image point
intensities of tiny objects are defined
primarily by the collection pathway, in CSLM the
illumination and detection pathways are equally
important to defining this spot that is imaged.
Both pathways travel through the objective lens
and the confocal scanhead.
X
Y
Z not shown (height is intensity)
sample
3
Where do the excitation and emission filters go?
Where is the confocal iris?
4
Illumination and detection path in MRC1024
confocal

• gas, KrAr laser

PMT detector
optical filters (Neutral Density and excitation ?)
confocal iris
optical fiber to carry laser to scanhead
emission ? filter
dichroic mirror (beam splitter) split excitation
from emission )
dichroic mirror
scan mirrors (galvo driven x y)
scan mirrors
objective lens
objective lens
sample
sample
EM
EX
5
Different light sources have different spectra
488nm
568nm
647nm
This is just one of the Hg lines at much higher
spectral resolution.
Low pressure gasses emit discreet lines of color
as in this low pressure Hg spectrum (grey)
overlaid with 3 of the major lines from a KrAr
mixed gas laser, also low pressure (color).
6
gas
gas
black body
gas
Metal halide are very similar to gas arc but
include metal iodides and bromides in the gas
mix. Longer life and tunable spectrum (based on
the metal used).
black body
All adapted from Murphy 2001
High pressure gasses in arc lamps emit relatively
broad spectrum light (many more vibrational
energy states than low pressure). Black body
radiation as emitted from the tungsten filament
in halogen/tungsten or standard tungsten filament
sources varies with filament temperature and are
UV and blue poor as well as being inefficient
(heat).
7
Neutral Density Filtering (all ? are affected
equally)
Beer / Lambert relationship
Transmittance T P / P0 Transmittance T
100 T
A-log T A  log10 P0 / PA  log10 1 / T
P0
Absorbance (OD)
P
A  e (pathlength) concentration e depends on
the filter material, pathlength is thickness of
filter, concentration is the conc. of material
with e in the filter
Lets say this filter blocks 70 of the incident
light so its T.30 So, this filter has an
A.125 What happens if we double the width of the
filter? T ? A? What is the A if the filter
blocks out 99.7 of the incident light?
8
Short and long pass filtering with colored
(colloidal) glass filters these do not affect
all wavelengths equally
Absorbance lt515nm
Alt515-log Tlt515
515 LP or OG 515
9
Filtering with interference filters (most but not
all band pass filters are interference filters)
All of our confocal band pass filters and
dichroic mirrors are interference filters, see
website below or Slayter Slayter 4.3. Also see
the optical thin film example on course website.
These filters are given absorbance and OD values
but they DO NOT follow the Beer Lambert
relationship with thickness and concentration.
http//hyperphysics.phy-astr.gsu.edu/hbase/hph.htm
l
10
Detector
Signal in
Signal out
DM
sample on microscope
Laser
Emission light (pink color) follows the same path
as the excitation light (red) until we reach the
dichroic mirror (DM). This DM prevents reflected
laser signal from causing decreased contrast of
our fluorescence signal.
Purdue Univ.
11
Many lens elements with complex shapes are needed
to bend and fold the light so that rays of
different colors and those closer or further from
the optical axis are focused to the same place.
When the objective lens is perfectly designed and
built, only then do we achieve diffraction
limited resolution as defined by the next slide.

These objective lenses are corrected for
chromatic and spherical aberrations and curvature
of field. Apochromats are corrected at more
wavelengths (colors) than achromats.
Murphy 2001
12
Gaussian image points vs. Abbes theory of image
formation Gaussian ray tracing brings rays to an
infinitely small and unacheivable point in space
(what is dmin). Abbes theory of image formation
states that a small interference pattern is
formed by the perfect lens, this is the
diffraction limited resolution. If you can
develop technology that elegantly overcomes this
barrier, you could win the Nobel prize.
dmin 1.22(?) / NAcond NAobj dmin 1.22(?) /
2 sin a ? dmin .61(?) / NAobj (for epi-
microscopy)NA (numericalaperture) sin a ?
Gaussian theory works well for image formation
of features larger than the dmin.
Abbes theory works for describing features that
are approximately the size of dmin or the
wavelength of light (or electrons) used.
13
What does the confocal iris or pinhole do?
Airy pattern with central disk.
Iris and diaphragm
This is a 2D view of a 3D phenomenon.
An interference pattern like this is projected at
the PMT for each object/ image point as the beam
scans the sample. The confocal iris or pinhole
selects only part of this Airy pattern to reach
the PMT, this can improve resolution. Its effect
on the z-axis diffraction pattern (not shown) is
even more dramatic. This is why with confocal we
can take optical z-sections.
Murphy 2001
14
Y
X
Z
From Pawley, 1995
15
Total voltage drop in a PMT may equal thousands
of volts.
Photocathode, usually coated, must be sensitive
to Einsteins photoelectric effect.
This PMT detector gives no spatial information on
its own, only gives counts. The eye does give
spatial information directly. It has an array of
detectors (neurons). A ccd or film camera also
directly record spatial information.
Eye is most sensitive to green light, PMTs can
have photocathodes made of materials sensitive to
specific colors (Murphy spectra p.25 )
http//www.olympusmicro.com/primer/flash/photomult
iplier/index.html
16
Now lets include system noise into the detector.
Lets assume that we get the equivalent of 1
photon/second of noise due to a light leak
(this could also be electronic noise). Lets
also assume that we get 16 photons/sec in real
signal. Our PMT converts each photon into 10
electronic counts (gain of 10). Given the
above information, what is the signal to noise
(S/N) ratio (in electronic counts) per pixel in
two cases 1-scan rate 1 pixel/second 2-
scan rate 1 pixel/10seconds? This can also
be called signal/background ratio. One step
further, all detector systems have some
inherent ability to detect differences in
intensity. It is not the signal /noise ratio
that is important here, it is the absolute
difference in signal intensity between 2 sample
points. Try this with this 4x4 pixel, 8 bit
image. Which pairs of pixels can be
differentiated from each other? Each pixel pair
has the same signal / signal ratio but the
absolute differences vary.
When sampling with more pixels, adjust scan rate
to increase photons/pixel this is a good idea
unless you are worried about beam damage! The
top row of 3 scans (not in box) was done at
constant scan rate (what is the scan rate for
the top row?) The photon flux from the sample
in all cases is 16 photons/second.
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