Light  microscopy

Light  microscopy    

Part  II  

What  is  numerical  aperature  (NA)?  

Usually,  higher  magnifica>on  objec>ves  have  greater  NAs  

Sample  specifica>ons  

objective magnification NA working distance (mm)

Achromat 10x 0.25 6.1 Achromat 40x 0.65 0.45 Pl Apo 100x (0il) 1.4 0.10

Working  distance  =separa>on  between  top  of  coverslip  and  front  element   of  objec>ve  when  specimen  is  in  focus  

Resolu>on  

Airy  Disk  Forma>on  by  Finite  Objec>ve  Aperture  

The  width  of     central  maximum   prop.  to  λ  and   inversly  prop.  to   objec>ve  aperature  

Lateral  Resolu>on  in  Fluorescence  Depends  on  Resolving   Overlapping   “ Airy  Disks ”  

Rayleigh Criteria: Overlap by r’, then dip in middle is 26% below Peak intensity

(2πx/λ)NA_obj

E.D.Salmon  

Minimum  resolvable  distance,  d _min  

Fluorescence:  d

 =  0.61λ/NA

[self-­‐luminous  object]  

Trans-­‐Illumina>on:  d

 =  λ/(NA

+  NA

)  

[note  that  resolu>on  depends  on  condenser  NA  too:  for  maximum     resolu>on  NA_cond  should  equal  or  exceed  NA_obj]  

Why  oil  immersion  lenses  provide  greater  resolu>on:  

they  have  a  larger  NA  (=nsin α )  

Resolu>on  is  be[er  at  shorter  wavelengths:  higher   objec>ve  NA  and/or  higher  condenser  NA  

High  NA  and/or  shorter  λ                                          Low  NA  and/or  longer  λ  

E.D.  Salmon  

Rayleigh  Criterion  for  the  resolu>on  of  two  adjacent  spots:  

 d_lim  =  0.61  λ_o  /  NA_obj    

Examples:    (λ_o  =  550  nm)    

 Mag  f(mm)        n    α    NA  d_lim  (µm)        (NA_cond=NA_obj)    high  dry    10x  16  1.00  15  0.25  1.10  

 40x  4  1.00  40  0.65  0.42   oil    100x  1.6  1.52  61  1.33  0.204  

 63x  2.5  1.52  67.5  1.40  0.196  

For  dry  objec>ves  NA  <  0.95;  for  oil  objec>ves  NA  <  1.52  with  oil  of  n=1.52  

Depth  of  field  (ver>cal)  resolu>on    D  =  0.61  λ  cos  α  /  n(NA)  

Low  power,  NA~  0.25                          D~  8  µm   Hi,  dry,                NA~0.5                                D~  2  µm   Oil  immersion,  NA~  1.3                    D~0.4  µm  

Higher  NA  means:  

Brighter  image  ~NA

Greater  lateral  resolu>on  

  Smaller  depth  of  field  

Contrast  

All  the  resolu>on  in  the  world  won ’ t  do   you  any  good,  if  there  is  no  contrast    

to  visualize  the  specimen.  

Contrast  

1 2 3 4 5 6 7 8 9 10

CONTRAST = (Isp - Ibg)/Ibg

HIGH LOW

E.D.Salmon  

diI-C₁₆

Thy-1 H-2 HA

Phase  contrast  microscopy  

Ridges  in  The  Surface  of  Cheek  Cells     for  Resolu>on  Tests  

High  Resolu>on  DIC  Microscopy

Keratocyte

Differential Interference Contrast (DIC) microscopy (from a goldfish scale, 3 times real time)

From  Ted  Salmon  

Walker  et  al,  Nature  347:  780-­‐782  

Dark  field  microscopy  

Interference  reflec>on  microscopy  (IRM)  

Illumina>on  for  the  microscope  

Purpose  of  Koehler  Illumina>on  

•  To  obtain  even  specimen  illumina>on  for   photomicrography,  video  microscopy  etc.  

•  To  use  field  diaphragm  alone  to  control   illuminated  area  of  specimen.  

•  To  control  the  angle  of  the  cone  of  

illumina>on(contrast  and  resolu>on)  by  

varying  condenser  diaphragm.  

A  Lamp  Collector  Lens  and  Microscope  Condenser  

Lens  are  Used  to  Concentrate  Light  on  the  Specimen  

Op>cal  Principle  

Summary  of  Köhler  Illumina>on  

•  Focus  specimen  at  low  magnifica>on  

•  Focus  and  center  field  diaphragm  by  adjus7ng  condenser   height  and  diaphragm  posi7on.  

•  Focus  lamp  filament  on  condenser  iris  diaphragm.  

•  Adjust  condenser  diaphragm  appropriately.  

–  For  visual  observa>on,  set  condenser  diaphragm  to     70-­‐90%  of  objec>ve  aperture.  

-­‐  To  enhance  contrast,  reduce  condenser  diaphragm  to  40-­‐50%  of   objec>ve  aperture.  

-­‐  For  video  microscopy,  set  condenser  aperture  to  ~objec>ve  aperture.  

Condenser  is  Translated  Along  Op>cal  Axis  to   Bring  Field  Diaphragm  into  Focus  

Condenser Focus Knob

Condenser X-Y

Translation Screws

Are Used to Center

Image of Field-

Diaphragm

Now, the field diaphragm controls the area illuminated on the specimen

Summary  of  Köhler  Illumina>on  

•  Focus  specimen  at  low  magnifica>on  

•  Focus  and  center  field  diaphragm  by  adjus>ng  condenser  height  and   diaphragm  posi>on.  

•  Focus  lamp  filament  on  condenser  iris  diaphragm.  

•  Adjust  condenser  diaphragm  appropriately.  

–  For  visual  observa7on,  set  condenser  diaphragm  to      70-­‐90%  of  objec7ve  aperature.  

-­‐  To  enhance  contrast,  reduce  condenser  diaphragm  to  40-­‐50%  of  objec7ve   aperature.  

-­‐  For  video  microscopy,  set  condenser  aperature  to  ~objec7ve  aperature.  

The  Condenser  Diaphragm  Controls  the   Illumina>on  NA  

An image of the Condenser Diaphragm is in-focus in the Objective Back Focal Plan (Aperture). As the condenser diaphragm is opened, the illumination NA increases without changing the area of specimen Illuminated (area controlled by Field Diaphragm).

Condenser and Objective Apertures

θobm Qcond

CD Cond SP OBJ OB FFP BFP

 A  prac>cal  note:  cleaning  

microscope  op>cs  

Effect  of  dirty  op>cs…..  

Taking  care  of  microscope  op7cs  

•  Never   “ dry ”  clean  a  lens  

•  Use  a  solvent  like  Windex  that  will  remove  most  everything.  

Use  xylene  under  a  hood  as  last  resort.  

•  Use  best  quality  lens  >ssue  available  [e.g  Kodak].  

•  Clean  in  swirl  pa[ern  from  center  out.  

•  Remove  immersion  oil  aqer  use  to    prevent  seepage  

Diatom Resolution

Test Specimens