leica dm ire2 microscope manual

LEICA DM IRE2 MICROSCOPE MANUAL
Neuroscience Imaging Core
Rightmire Hall
Ohio State University
Director: Tony Brown
Rightmire 060
292-1205
brown.2302@osu.edu
Facility Manager: Paula Monsma
Rightmire 062
292-3025
monsma.1@osu.edu
This manual prepared by Tony Brown.
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TABLE OF CONTENTS
LEICA DM IRE2 MICROSCOPE MANUAL ................................ 1
Getting to know the microscope .............................................. 3
Microscope control panel ......................................................... 7
LCD display panel ...................................................................... 8
LCD display panel buttons ....................................................... 9
Changing filters ....................................................................... 10
Changing objectives................................................................ 11
To switch between dry and immersion objectives ............... 12
Adjust halogen lamp brightness ............................................ 13
To focus using the focusing knobs ....................................... 14
Using the focusing buttons .................................................... 15
Changing the coarseness of the focusing ............................ 16
Eyepieces ................................................................................. 17
Placing a slide on the Z-galvo stage...................................... 17
Transmitted light detector selection knob ............................ 18
Bright field observation .......................................................... 18
Koehler illumination ................................................................ 19
Phase contrast ......................................................................... 20
Differential interference contrast (DIC) ................................. 21
Switching stage holders ......................................................... 22
Using immersion objectives ................................................... 23
Cleaning objectives ................................................................. 24
Applying oil to objective without removing slide ................. 25
Specimen preparation ............................................................. 26
DOs and DON’Ts ...................................................................... 27
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Getting to know the microscope
VIEW FROM LEFT
Transmitted light
illumination column
(tilts back for better
access to stage)
Transmitted
light detector
Eyepieces
Condenser
lens
Z- galvo stage
(detachable)
Filter cube
turret
Laser scan
head
with 3
fluorescence
detectors
Side port
Focus knob
Halogen lamp
intensity
adjustment
wheel
3
Microscope
control panel
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VIEW FROM RIGHT
Halogen lamp
housing
(transmitted light)
Filter holders
(empty)
Transmitted light
detector
selection knob
Condenser turret
Mercury lamp
housing
(epi-fluorescence)
Objective turret
Tube lens module
with Bertrand lens
Focus knob
X-Y stage
movement
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OBJECTIVE TURRET (DETAILED VIEW)
objective
prism turret
objective
turret
Epifluorescence
Illumination
diaphragm
(controls
brightness of
illumination)
Epi-fluorescence
field diaphragm
(controls area of
illumination)
filter cube
turret
cuvesfou
5
DIC analyzer
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OBJECTIVE PRISM TURRET POSITIONS
Objective
turret
position
Description
Matching objectives
BF
Empty (for bright field
or phase contrast)
C
DIC objective prism
D
DIC objective prism
E
DIC objective prism
20x multi-immersion
63x water
immersion
100x oil immersion
40x oil immersion
63x oil immersion
CONDENSER TURRET POSITIONS
Condenser
turret
position
Description
Matching objectives
BF
Empty (for bright
field)
PH1
phase ring
10x dry
PH2
phase ring
40x dry
20
40/63
63/100
DIC condenser
prism
DIC condenser
prism
DIC condenser
prism
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20x mulit-immersion
40x oil immersion
63x water immersion
63x oil immersion
100x oil immersion
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Microscope control panel
LCD display
LCD display
panel buttons
Red light
shows current
filter selection
switch filter cube
GFP: green fluorochromes
RED: red fluorochromes
CFP/: far red fluorochromes
SCAN=empty slot
select port
VIS=light to eyepieces
SIDE=light to scanner
BOTTOM: disabled
Mercury lamp
shutter
(red light is on
when shutter is
closed)
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switch tube lens
(disabled)
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LCD display panel
 arrow
indicates
lower limit
has been
set
Indicates
current
step size
setting for
focusing
Indicates
position of
objective
relative to
upper limit
Indicates
current
objective
Indicates
desired DIC
objective
prism
 arrow
indicates
upper limit
has been
set
Indicates
current filter
cube
D= dry mode
I= immersion mode
Indicates
actual DIC
objective
prism in light
path
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LCD display panel buttons
Learn mode
(DO NOT
USE!)
Toggle
between
voltage
readout
and
objective
readout
Set lower
limit of
travel for
objective
(DO NOT
USE!)
Set upper
limit of
travel for
objective
(DO NOT
USE!)
Adjust step
size for
coarseness
of focusing
Do not press the LEARN button (this will enter you
into learn mode, which you must not play with). If you
accidentally press this button, you will see the word
“EXIT “ flashing. Press the LEARN button again to
exit the learn mode.
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Changing filters
Use the motorized fluorescent filter cube changer on
the microscope control panel:
Press left button
to rotate filter
cube turret left
Press right button
to rotate filter
cube turret right
Red light shows current filter cube selection
The filter cube turret contains three filters plus an
empty slot
 GFP : cube for green fluorochromes
 RED : cube for red fluorochromes
 CFP/ : cube for far red fluorochromes
or cube for CFP (see Paula for details)
 SCAN : empty slot
Notes:
 The cube in the CFP/ position is normally the far
red cube, suitable for fluorochromes such as
TOTO-3 and Cy5
 The SCAN position is used for confocal imaging
and for transmitted light observation through the
eyepieces
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Changing objectives
Use the objective turret control buttons on the left
side of the microscope
Press upper
key to
increase
magnification
Press lower
key to
decrease
magnification
Current objective indicated on LCD display panel
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To switch between dry and immersion objectives
The standard objectives on our microscope are
grouped into three blocks or modes:
100x oil immersion
63x oil immersion
oil immersion mode
40x oil immersion
20x oil/water/glycerol immersion
40x dry
10x dry
multi immersion mode
dry mode
To reduce the chance of immersion medium getting
on to a dry objective or the chance of mixing of
different immersion media, the microscope will not
allow you to move freely between these modes using
the objective turret control buttons.
To switch from one mode to another:
 simultaneously press the “upper limit” and “lower
limit” buttons on the microscope control panel
 the words “CHANGE OBJECTIVE” flash on the
LCD display panel
 now you can change the objective using the
objective turret control buttons
Press these two
buttons
simultaneously
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Adjust halogen lamp brightness
Use the dial on the front left side of the microscope
stand near the base
Halogen lamp
brightness
control
The lamp voltage will display automatically on the
microscope control panel display when the intensity
dial is adjusted
To switch off transmitted light illumination adjust
lamp intensity to 2.5V then continue rotating dial
beyond this point
0V on control panel display indicates illumination is off
To switch on transmitted light illumination, rotate
briefly in the opposite direction
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To focus using the focusing knobs
One way to focus is using the focusing knobs
located on the left and right side of the microscope.
Turning the knob so that your thumb moves away
from you focuses down
Turning the knob so that your thumb moves toward
from you focuses up
Focusing knobs
(turn in direction of arrows to
focus objective down)
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Using the focusing buttons
Another way to focus is to use the focusing buttons
located on the right side of the microscope
Press upper
focus key to
focus up
Press lower
focus key to
focus down
Note about upper limits:
If an upper limit is set for the objective (see LCD
display panel) then the objective will not move above
that limit if you are focusing using the focusing
buttons.
The only way to focus above the upper limit is to use
the focusing knobs
This is a safety feature to prevent accidental
damage of the objective when focusing using the
focusing buttons.
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Changing the coarseness of the focusing
The focusing is electronic and has five settings:
Setting
Step size
S0
S1
Fine
Medium fine
S2
Medium coarse
S3
Coarse
You can use any step size with any objective, but
when you first select an objective the default step
size will be as follows:
Objective
Setting
63,100x
40x
S0
S1
20x
S2
10x
S3
Press STEP button to switch between S0, S1, S2
and S3
STEP button
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Eyepieces
Adjusting interpupillary distance:
 Adjust eyepieces to match interpupillary distance
by moving the eyepieces closer together or further
apart
Adjusting parfocality
 Focus on specimen using electronic focusing
controls
 Close right eye and adjust the left eyepiece so that
the image appears in focus to your left eye
 Close the left eye and adjust the right eyepiece so
that the image appears in focus to your right eye
 The eyepieces are now parfocal
Placing a slide on the Z-galvo stage

To avoid touching or damaging the objective,
lower the objective turret all the way using the
focusing buttons
2



2
1
1
1
Position the slide holder clips as shown above
Insert slide into the holder in a front-to-back
motion (1)
Slide clips inward onto slide to secure the slide (2)
1
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Transmitted light detector selection knob
For any transmitted light
observation (bright field, DIC,
phase contrast), this knob should
be in the vis position.
Bright field observation
Bright field observation means observation with
transmitted light using no contrast enhancement
method (e.g. no phase contrast or DIC)
For bright field observation:
 rotate condenser turret to BF (empty) position
 rotate objective prism turret to BF (empty)
position
 switch filter cube turret to SCAN (empty) position
Condenser turret in bright
field (BF) position
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Koehler illumination
Koehler illumination is essential to obtain good
transmitted light images








Select objective
Open the condenser aperture diaphragm
(move lever to the right)
Focus on specimen
Close the field aperture diaphragm
(move lever to left)
Focus condenser using condenser focusing
knob until the image of the aperture is sharp
If necessary center the field diaphragm in the field
of view using the two centering screws located
on the front of the condenser
Open field aperture diaphragm until it just
disappears from the field of view
Close down the condenser aperture diaphragm
until the desired contrast is achieved
Field aperture
diaphragm
centering
screws
Condenser
focusing knob
DIC polarizer
Condenser
aperture
diaphragm
Condenser
turret
Phase ring centering
keys insert here
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Phase contrast
For phase contrast, you need:
 a phase contrast objective
 a matching phase ring in the condenser turret
To set up phase contrast:
 Select a phase contrast objective (i.e. either of the
dry objectives)
 rotate objective prism turret to BF (empty)
position
 Rotate condenser turret to select phase ring
PH1 for 10x
PH2 for 40x
 Focus on specimen
 Insert the Bertrand lens into the optical path
using by rotating the tube lens module from
“SCAN” to “B”
 Focus on the phase ring using the Bertrand lens
focus slider on the tube lens module
 If condenser phase ring (dark ring) is not centered
on the objective phase ring (light ring), center it
using the insertable phase ring centering keys
(see Paula for instructions)
 Set Koehler illumination
Tube lens
module in B
position
Bertrand lens
focus slider
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Differential interference contrast (DIC)
For DIC, you need:
 A DIC objective (i.e. any of the immersion
objectives)
 A polarizer above the condenser (no need to
insert this – it is kept in the light path at all times)
 A condenser prism in the condenser turret
 An analyzer beneath the objective prism turret
 An objective prism in the objective prism turret
To set up DIC:
 Select a DIC objective (any of the immersion
objectives)
 The objective DIC prism required for that objective
is displayed on the microscope control panel
 Manually rotate condenser turret to select the
appropriate objective DIC prism
Condenser
prism
63/100
63/100
Objective
prism
D
E
63x water
40/63
D
40x
20x multi-immersion
40/63
20
E
C
Objective
100x oil
63x oil


Manually rotate objective prism turret to select
objective prism
Insert analyzer into light path
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Switching stage holders
The microscope has two stage holders:
 Z- galvo stage (permits rapid and precise
movement in Z axis for acquisition of Z stacks)
 Universal stage holder (holds a wider range of
dishes)
To remove Z-galvo stage:
 Unscrew the two thumbail screws (circled in red):

Place stage (still attached to the cable) on top of
the laser scan head as shown:
Take care not to touch condenser lens surface when
removing and replacing the stages!
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Using immersion objectives
OIL immersion:
 Clean coverslips before using immersion
objectives
 Apply oil to the objective or to the coverslip before
placing your slide on the stage
 Use only LEICA IMMERSION OIL
 Use the minimum amount of oil necessary
 If you use too much oil it may run down the side of
the objective and damage the optics!
To apply oil to an objective:
 dip oil applicator in immersion oil bottle
 allow excess to drain off
 apply oil to objective by touching to the metal next
to the lens
 DO NOT TOUCH THE LENS DIRECTLY!
WATER immersion:
The 63x water immersion objective (not normally
installed on the microscope) uses water rather than oil
as the immersion medium.
Contact Paula for instructions on using this objective.
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Cleaning objectives
This microscope is equipped with the highest quality
objectives, with a total value exceeding $30,000!
You must exercise great care to preserve these
objectives
To clean oil off immersion objectives:
 Remove specimen from stage
 Blot (not wipe!) off excess oil using lens tissue
 Use a fresh area of the lens tissue for each blot
 Repeat blotting until no more oil comes off onto
the lens tissue
 Wipe the metal housing around the lens using lens
tissue
 It typically takes 2 or 3 sheets of lens tissue to
properly clean a single objective
What to do if you accidentally get oil on a dry
objective:
 Blot off excess with lens tissue
 Then contact Paula, who will clean the lens with
special cleaning fluid (do not attempt this yourself!)
 Lens tissue alone will not get all the oil off the lens
and this will interfere with the optics
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Applying oil to objective without removing slide
FOR ADVANCED USERS ONLY!
You can use the following procedure if you are
observing your slide and you wish to switch to an oil
immersion objective without removing the slide from
the stage holder.
 Focus objective turret all the way down using the
focus down button
 In the LCS software, click on obj button and
select the objective diametrically opposite in the
objective turret from the objective you want to
apply oil to (i.e. 3 positions away from the
current objective)





Objective you want to
use
100x oil
63x oil
Objective in opposite
position
20x multi-immersion
40x dry
40x oil
10x dry
20x multi-immersion
40x dry
10x dry
100x oil
63x oil
40x oil
This will rotate the objective that you want to apply
oil to so that it is accessible from the right of the
microscope
Apply oil to the objective
In the LCS software, click on obj button and
select the objective that you applied oil to
This will rotate the objective back into the light
path
Focus up on your specimen
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Specimen preparation
Some general tips:
 Select fluorochromes that are optimally excited by
the confocal laser lines
 For multiple labeling, the less overlap between the
excitation and emission spectra the better
 Always use #1.5 coverslips
 Mount coverslip to slide securely and seal with nail
enamel or mounting medium that solidifies
 Do not observe on the microscope until the nail
enamel or mounting medium has hardened!
 Secure the slide with the clips when acquiring Z
series
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DOs and DON’Ts
DOs
 Use only lens tissue to clean objectives
 Use Kimwipes to clean coverslips and slides
DONTs
 Never touch any optical surface with anything
other than lens tissue
 Never clean the objectives with any kind of solvent
 Never use Kimwipes to clean objectives or any
other optics
 Never use any liquid cleaners or solvents to clean
the microscope optics or any part of the
microscope!
When to ask for Paula’s assistance









Changing mercury or halogen bulbs
Aligning mercury and halogen lamps
Adding or removing objectives
Adding or removing filter cubes
Using the 63x water immersion objective
Cleaning oil off 10x and 40x dry objectives
Using the Leica objective warmer
Using the Bioptechs heated chamber
Any other time you are unsure what you are
doing!
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Extras
Setting
Step size
Objective
S0
S1
Finest
63,100x
40x
S2
20x
S3
SC
10x
Coarsest
Note about the coarsest focusing setting (SC):
Press both upper and lower focus keys
simultaneously to switch to the coarsest focusing
setting (SC)
Press both focus keys simultaneously to switch back
to fine focusing setting (S0-S3)
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