How to Choose the Optimal Objective?

Home , Apochromat, Oil immersion

How to choose the optimal objective?

Dr. Sebastian Gliem CARL ZEISS Microscopy Embedded Specialist, HCBI

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 1 AGENDA

I. Importance

II. Types/Tasks

III. Properties

IV. How to choose

V. Choices

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 2 Why to think about objective selection?

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 3 Why to think about the right objective?

• Challenging applications in Life Cell Microscopy • Variety of methods in Life Cell Microscopy

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 4 Challenging Applications

Demanding applications in light microscopy of living cells are

• high- resolution microscopy of very thick samples

• high- resolution microscopy of structures remote from the cover glass

• high- resolution microscopy of fast moving structures

Usually these applications are linked to fluorescence microscopical methods!

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 5 Method Approaches in Living Cell Microscopy

Optical Methods applied to achieve perfect images in demanding fluorescence applications are Sectioning results in • Optical sectioning with structured illumination more (e.g. ApoTome) Information • Confocal pinhole techniques (e.g. Confocal Laser systems, Spinning Disc systems)

• Evanescent fields (TIRF)

• Single plane illumination (LSFM)

• Mathematical approaches (e.g. 3D/ 2D Deconvolution) Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 6

Why to think about the right objective?

• Challenging applications in Life Cell Microscopy • Method approaches in Life Cell Microscopy • Sample preparation, e.g. immersion • Sample properties, e.g. sample size

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 7 Influences of the sample preparation

Immersion • Acquired with clean lens • Wrong immersion • Always use a clean objective! • Acquired with old oil remnants

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 9 Sample Influence: Wrong Refractive Index

A wrong immersion medium with The use of our proprietary CARL ZEISS immersion media is a a deviant refractive index and/ or prerequisite in live cell imaging dispersion will introduce spherical and chromatical aberration to the image For optimum results: Oil immersion systems with IMMERSOL TM 518 F Examples: Water immersion objectives with distilled water or IMMERSOL W •Using immersion oil with a water (artificial non- evaporating, low- viscosity „water“). A must for long- immersion type objective time experiments

•Applying low- viscosity immersion media (e.g. anisol) ALWAYS REMOVE OLD RESIDUES OF IMMERSION MEDIUM instead of immersion oil (e.g. FROM THE FRONT LENS. DO NOT MIX BATCHES TM IMMERSOL )

•Employing embedding media with a refractive behavior strongly deviant from immersion oil will add to an inferior signal to background noise ratio in fluorescence

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 10 Why to think about the right objective?

• Challenging applications in Life Cell Microscopy • Method approaches in Life Cell Microscopy • Sample preparation, e.g. background staining • Sample properties, e.g. sample size

• Different types of objectives • Different objective properties • Different objective corrections

 The Perfect Microscopical Image

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 11 The Perfect Microscopical Image

The perfect microscopical image

The • has a magnification that matches with a given structure size Microscope Objective is • is of maximum possible detail rendition in x,y Responsible and z for the • has the highest possible contrast Formation of a Perfect Image

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 12 Types of objectives

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 13 Types of objectives (selection)

Ultrafluar A-Plan Fluar Achroplan C-Apochromat

Plan-Neofluar

LD Plan-Neofluar Apochromat EC Plan-Neofluar

LD LCI Plan-Apochromat Plan-Apochromat

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 14 Types of objectives (selection)

Main differences Consequences

Glass material  Light transmission efficiency (λ)

 Light ray Amount of lenses representation in your image (= degree of Shape of the glass correction)

Glass coatings

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 15 The Objective Colour Code

Today, for ease of use, all microscope objectives follow a colour code that allows immediate recognition of important objective parameters

The standard colour code of objectives was introduced to microscopy in 1953 by Dr. Kurt Michel at CARL ZEISS in Göttingen

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 16 Tasks of objectives

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 18 Tasks of objectives

The tasks of microscope objectives

• magnify (M) the image structure The microscope • resolve (n.A.) the image structure objective is • offer inter-sample-objective correction the most capabilities (e.g. immersion, corr-ring) important • provide a necessary working distance optical • avoid blurred edges (spherical aberration) component • image different colors in one point for (chromatic aberration) imaging • high light transmission for required λ

• applicable for wanted contrast techniques (BF, DF, Ph, DIC, Pol, Fl)

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 19 Properties of objectives

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 20 Objective properties: Resolution and numerical Aperture

The resolution of a microscopical image depends on the actual numerical aperture (n. A.) of the given objective/ and the wavelength of light used

λ λ d0 = = α 2 n. A. 2n * sin α α 1

The resolution formula of the microscope was developed in 1872 by Prof. Ernst Abbe at CARL ZEISS

In 1905 Dr. Moritz von Rohr at CARL ZEISS invented the first n. A. max Immersol ~ 1,46 objective with an n. A. = 1,68 (n. A. max Monobromnaphtalene ~ 1,68 Toxic!) (toxic immersion medium)

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 21 Objective properties: Resolution and numerical Aperture

The wider the opening angle of the objective (2) . the more of the diffracted light can be captured 2 . the smaller details (1) can be resolved α α 1 . n = refractive index of the medium between object and objective nair = 1, nglass = ~ 1.52 . a = half the opening angle of the objective

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 22 Objective properties: Differentiation between two points

What is the requirement that two points can be differentiated from Light wavefront each other?

When light originates from single points, it generates so-called diffraction patterns, that contain structural information

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 23 Objective properties: Differentiation between two points

When two diffraction patterns of 2 Objective neighbouring points 1 interfere with each Light wavefront 0 other they generate 1 interference maxima 2 that are captured by the objective (intermediate image)

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 24 Objective properties: Differentiation between two points

 To perceive two 2 Objective points as separate 1 points, the Light wavefront 0 objective needs to 1 collect at least the 2 1st order maximum of the diffraction interference pattern 1

Fine structure 0

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 25 Objective properties: Differentiation between two points

Principal maximum of Intensity object 1 (centre of Airy Disc) coincides with first 1 2 minimum of object 2: 2

 Minimum distance d0 is reached (limiting resolution)

 Rayleigh-criterion to achieve sufficient 1 2 X contrast: d0 Intensity of maxima 20% higher than intensity of minimum

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 26 Objective properties: Differentiation between two points

Theoretical maximal λ d0 = resolution d0 n. A.Objective + n. A.Condenser

Simplified formula (wo λ d = condensor) for resolution d 0 0 2 n. A.Objective

1.22 x λ Maximal resolution d0 in d0 = reality 2 n. A.Objective

Example Green light λ = 550 nm, n. A. = 1.4 (Oil immersion)

d0 = 671 nm / (2 x 1.4) = 239 nm = 0.239 µm

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 27 Objective properties: Immersion enables higher n.A.

Immersion prevents light rays from being deflected between α1 α2 two materials, e.g. between objective objective front lens and coverglas immersion

α α  More order 1 2 coverglas maxima of the diffracted light pattern can be collected

 Higher resolution! n. A. max Air = 0,95 n. A. max Immersol/ Immersol HI ~ 1,46 - ~ 1,57 (n. A. max Monobromnaphtalene ~ 1,68 Toxic!)

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 28 Objective properties: Higher n.A. = More Brightness

Excitation A high n.A. objective α α illuminates the 1 2 sample with a larger cone of excitation Emission light and can also α1

α α capture a larger cone 1 2 of emission light

 Fluorescence brightness Example: theoretically growths with N Achroplan 40x/ 0,65 Dry (n.A.)4 vs EC Plan- NEOFLUAR 40x/ 1,30 Oil

The EC Plan- NEOFLUAR with the double n. A. value is 24 = 16x brighter!

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 30 Objective properties: Higher n.A. = More Brightness

CAVE

Next to n.A……

Additionally, glass properties and applied wavelength determine light transmission efficiency

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 31 Objective properties: Higher n.A. = Smaller depth of field

The depth of field is the z-thickness inside the object field that is imaged sharp together

The depth of field is increasing with the decrease of the n.A.

 (n.A.)2 ~ 1/ Depth of Field Example:

Depth of field α Plan-Apochromat 100x/1,46 = 0,23 µm

Depth of field Plan-Apochromat 20x/0,8 = 1,32 µm

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 32 Objective properties: Lens aberration

Types of lens Spherical aberrations aberration Chromatic aberration Curvature of the field Astigmatism Distortion Coma

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 33 Objective properties: Spherical aberration

Difference of the focal point for rays 6 traveling at different 1 distances to the optic axis

 Blurred image  Compensated by aspherical lenses

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 34

Objective properties: Curvature of the field

Imaging through a curved lens surface causes a curved image plane

 Image with unsharp edges

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 36 Objective properties: Flatness of field (“Plan-Objectives”)

 Compensated by APOCHROMAT Flat Field >> 25!

steeply curved EC- Plan- NEOFLUAR Flat Field > 25! lens surfaces at the back of the W- N ACHROPLAN Flat Field ~ 20 objectives and by a concave F- FLUAR Flat Field ~ 17 meniscus within FLUAR (non flattened!) Flat Field ~ 14 the front lens

 “Plan-” correction invented by CARL ZEISS in 1938

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 37

Objective properties: Chromatic aberration

Objectives have color artefacts (e.g. color fringes) in x/y and z

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 38 Objective properties: Chromatic aberration

 Compensated by CARL ZEISS APOCHROMATs are fully colour corrected no a combination of longer for only 3 – 4 spectral lines unique glasses with different color refractive properties

 “Apochromatic-” correction invented by CARL ZEISS in 1886

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 39

Objective properties: Chromatic aberration

 Compensated by CARL ZEISS APOCHROMATs are fully colour corrected no longer for only 3 – 4 spectral lines, but for a full spectral a combination of range (this corresponds to a correction of up to 14 (!) spectral unique glasses lines on the „old scale“) with different color refractive e.g. C- APOCHROMAT 40x/ 1.2 W Korr UV- VIS- IR is fully properties colour corrected from ~ 365 to ~ 900 nm

 “Apochromatic-” correction invented by CARL ZEISS in 1886

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 40

How to choose the right objective

Carl Zeiss Microscopy GmbH, Sebastian Gliem, HCBI 01.08.2016 Seite 42 Objective choice: Magnification and Resolution