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 min = 0.61λ/NA obj [self- luminous object] Trans- Illumina>on: d min = λ/(na obj + NA cond ) [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 (=nsinα)
E.D. Salmon 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
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 2 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 CONTRAST = (Isp - Ibg)/Ibg HIGH LOW 1 2 3 4 5 6 7 8 9 10 E.D.Salmon
Phase contrast microscopy dii-c 16 Thy-1 H-2 HA
Ridges in The Surface of Cheek Cells for Resolu>on Tests High Resolu>on DIC Microscopy E.D.Salmon
Keratocyte Differential Interference Contrast (DIC) microscopy (from a goldfish scale, 3 times real time)
Walker et al, Nature 347: 780-782 From Ted Salmon
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 Condenser and Objective Apertures Q cond θ ob m CD Cond SP OBJ OB FFP BFP 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).
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