Light microscopy. Part II
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1 Light microscopy Part II
2 What is numerical aperature (NA)?
3 Usually, higher magnifica>on objec>ves have greater NAs
4 Sample specifica>ons objective magnification NA working distance (mm) Achromat 10x Achromat 40x Pl Apo 100x (0il) Working distance =separa>on between top of coverslip and front element of objec>ve when specimen is in focus
5 Resolu>on
6 Airy Disk Forma>on by Finite Objec>ve Aperture The width of central maximum prop. to λ and inversly prop. to objec>ve aperature
7 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
8 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 ]
9 Why oil immersion lenses provide greater resolu>on: they have a larger NA (=nsinα)
10 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
11 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 x oil 100x x For dry objec>ves NA < 0.95; for oil objec>ves NA < 1.52 with oil of n=1.52
12 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
13 Higher NA means: Brighter image ~NA 2 Greater lateral resolu>on Smaller depth of field
14 Contrast All the resolu>on in the world won t do you any good, if there is no contrast to visualize the specimen.
15
16 Contrast CONTRAST = (Isp - Ibg)/Ibg HIGH LOW E.D.Salmon
17
18 Phase contrast microscopy dii-c 16 Thy-1 H-2 HA
19 Ridges in The Surface of Cheek Cells for Resolu>on Tests High Resolu>on DIC Microscopy E.D.Salmon
20 Keratocyte Differential Interference Contrast (DIC) microscopy (from a goldfish scale, 3 times real time)
21 Walker et al, Nature 347: From Ted Salmon
22 Dark field microscopy
23 Interference reflec>on microscopy (IRM)
24 Illumina>on for the microscope
25 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.
26 A Lamp Collector Lens and Microscope Condenser Lens are Used to Concentrate Light on the Specimen
27
28 Op>cal Principle
29 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.
30 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
31 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.
32 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).
33 A prac>cal note: cleaning microscope op>cs
34 Effect of dirty op>cs..
35 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
36
37 Diatom Resolution Test Specimens
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1/30/14 Reflection - wave hits non-absorptive surface surface of a smooth water pool - incident vs. reflected wave law of reflection - concept for all electromagnetic waves - wave theory: reflected back
Purpose: Explain the top 10 phenomena and concepts. BPP-1: Resolution and Depth of Focus (1.5X)
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