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Technical specifications for an absorption-fluorescence
microspectrophotometer dedicated to the study of fluorescent proteins in
microdrops or crystals for the Grenoble “Pixel” team
The microspectrophotometer of the Grenoble “Pixel” team (IBS/IRTSV) is dedicated to the
study of changes in protein absorption and fluorescence, both in microdrops and crystals.
Previous versions of the microspectrophotometer have been built and described (Bourgeois et
al., "A novel microspectrophotometer for absorption and fluorescence studies of protein
crystals " J. Appl. Cryst., (2002), 35, 319-326, Royant et al. (2007) "Advances in
spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements" J.
Appl. Cryst. (2007), 401105-1112.)
We are now willing to install a new instrument combining improved versatility, multiple
beam alignment capability and stability. This instrument is based on two objectives instead of
three in the previous versions.
The change in design from three to two objectives necessitates the simultaneous coupling of
multiple light sources/detectors into a single objective operating in back scattering mode.
Exchangeable coupling optics modules are inserted between an objective with infinity
correction and an on-axis illumination /video-visualisation system.
Key features remain: overall compactness, operation under cryogenic conditions (Oxford
series nitrogen cryojet) and maximisation of free space around the sample. In addition, a key
point is that the device should be easily upgradable to the possibility to perform high-quality
fluorescence imaging.
The microspectrometer consists of a goniometer-mounted sample holder, a pair of opposing
objectives, one of which (A) is directly fiber-coupled, whereas the other (F) is coupled to a
distribution stage that allows to simultaneously channel the in-/output from/to three different
fibers, and on-axis illumination and observation facilities (see schematic drawing below). The
(F) objective should be easily exchangeable via an adjustable revolver support.
video-observation
illumination
kohler
stage 3:
coupling to spectro F
stage 2:
coupling to laser 1
stage 1:
coupling to white lamp or laser 2
adjustable objective revolver
mirror objective with parallel output
xy
phi,z-gonio
with sample holder
xyz
mirror objectif fiber coupled
to spectro A or white lamp
granite
Schematic design
1. Optics for the spectrometer:
a) The optics of the spectrometer consist of 2 paired objectives facing each other with the
following specifications:
 In-/Output Connections from/to detector/light sources by 50 to 800 µm optical fibres
through SMA connectors for the A objective, parallel input/output from/into
distribution stage for the F objective.
 Objective-to-sample distance of at least 25 mm to avoid steric hindrance around the
sample.
 Overall magnification of objective +coupler should be in the range of 4 to 8.
 Numerical aperture ≥ 0.28
 Objectives should be designed to work with UV-visible light in the range 250nm750nm. Mirror objectives shall be considered to minimize chromatic aberrations over
this wavelength range. Overall transmission through a 30 µm diameter pinhole at the
sample position should be greater than 25% in the 400-750 nm range and greater than
10% in the 250-400 nm range (ratio of light intensity out of input fibre to light
intensity out of the output fibre, 100 m diameter fibres).
 Independent, lockable, X,Y,Z positional adjustments of the A objective and X,Y
positional adjustments of the sample holder should enable alignment of the goniometer
rotation axis (Z axis) with the two objectives with better than 5µm accuracy.
 Objectives should be equipped with 1 or preferably 2 slot(s) for 1 inch filters each
equipped with compensation plates if not in use and compatible with different types of
infinity corrected reflective or refractive objectives if in use.
 Objectives should be equipped with retractable rotating polarizers/analyzers.
(extinction coefficient better than 10-4 in the 350-650 nm range). Dedicated slots
should preferably be available to insert polarizers/analyzers.
b) Distribution stages
 Serve to separate the in/output of the infinity-corrected objective into three separate 50
to 800 µm optical fibers (to laser, white lamp, spectrometer, respectively) through
SMA connectors. Each module typically includes a dichroic mirror (DM) separating
input laser and output light to the spectrometer or a broadband beamsplitter (BS)
combining input laser and white light. The modules should additionally be equipped
with 2 slots for 1 inch filters each equipped with compensation plates if not in use.
 The three fibre coupled modules should be adjustable independently such as to ensure
that all three beams image the identical (within 5µm) sample area throughout the
wavelength range 250-750 nm (i.e. no chromatic abberation).
 1) Beamsplittter position (mixing laser and white light): 0/100, 30/70, 50/50,100/0 at
choice
 2) Dichroic mirrors(separating laser excitation from fluorescence detection): various
wavelength (405, 473-488, 532, 561 nm) + 0 and 100 %T at choice
2. Mechanical set up:
 The mechanics of the bench, and the chosen material should provide sufficient rigidity
and thermal stability in order to ensure sample positional accuracy of 5 m in X, Y, Z
relative to the objectives over 24 hours..
 The chosen materials (possibly with protective shields) should withstand temporary
exposure to liquid nitrogen.
 In order to avoid laser hazards, the surface of the bench must absorb UV-visible light.
 Also mounted onto the bench and adjustable in X, Y a one circle goniometer should be
inserted that holds the sample.
 Space symmetrical to the column relative to sample position should be kept free so as
to reserve space allowing for setting up various devices such as a Oxford series 600
cryogenic system and/or different sample controlling devices.
3. One circle goniometer
 The goniometer holding the sample (through a standard gioniometer head used in
protein crystallography) should consist of motorized rotation () and translation (Z)
stages controllable with joystick and Labview driver. The sphere of confusion over
360° rotation should not exceed 5 µm in diameter.
 The translation stage should have at least 20 mm travel range with 5 µm accuracy and
1 µm repeatability.
 The rotation stage should rotate with better than 0.2° accuracy and repeatability.
4. Sample visualisation
 The sample should be visualized through a video-microscope coupled to a CCD colour
camera. An objective, preferably with zooming capability (field of view on CCD of
the order of H 0,46 x V 0,34 mm at full zoom to H 5,5 x 4,1 mm at minimum zoom)
should allow sample centering..
The observation objective should be placed in the same objective revolver as the
infinity corrected objective.
Sample illumination should preferably be realized with a standard lamp in Kohler
mode. A filter slot to cut out residual UV light should be available. Alternatively, an
independent sample illumination source could be envisaged.
5. Fluorescence Imaging
The device should be compatible or easily upgradable to the possibility to perform
fluorescence imaging with, e.g., a high NA Mitutuyo objective with long working distance
(mountable on the observation objective revolver, 95 mm parfocal distance) and a dedicated
module replacing one of the described stages and connected to an EMCCD camera.