Diagnosing Microscope Drift Problems

Introduction

This technical note describes how to diagnose and fix problems with drift in timelapse imaging. Drift occurs when the XY position or Z focus of the sample moves outside of software control.

Note that this note does not cover repeatability. Repeatability is the ability of an XY stage or Z drive to return to the same position when moved between locations. Drift is movement in X, Y or Z when the sample should be static.

Quantify the Drift

To quantify the drift:

• Put a fixed sample on the microscope (e.g. a calibration slide, calibration beads or a fixed specimen)
• Ensure that the system is spatially calibrated (this is because we want to discover how far the drift is in µm rather than in pixels)
• Image the fixed sample to acquire a timelapse over a period that shows the drift

The timelapse image will show the direction and speed of the drift over time. This experiment should be repeated after changing each factor that could cause drift to quantify the effect of any changes.

Temperature

Most drift problems are caused by temperature variations. As the microscope and stage heat up or cool down, they expand and contract. To get stable timelapse imaging, it is essential to ensure the temperature of the whole microscope is constant. The level of drift from temperature will vary with the model of microscope, but drifts of more than 1 µm/?C in X, Y and Z are not uncommon.

Symptoms of temperature-related drift can include:

• Drift occurs first in one direction and then in the reverse direction as the microscope warms and cools
• Drift is less in the afternoon (when the system is warm) than in the morning (when it is cold but heating up)
• Drift starts quickly but then gradually stops (as the temperature of the microscope stabilises)

The temperature of the microscope can be affected by several factors:

• The ambient temperature in the room
• Heating caused by HBO lamps, TL lamps and other light sources
• Heating caused by electronics inside the microscope body
• Heating caused by incubation units and stage or objective heaters

To avoid thermal drift, it is essential that the temperature remains stableShort-term fluctuations in temperature (such as a blast of cold air from an opened door) will not matter too much as the mass of the microscope body will not change temperature very quickly. What is critical is the long-term average temperature of the system.

To solve thermal drift problems:

• Ensure that the temperature in the room is stable to within +/- 1?C. Use an inexpensive min/max digital thermometer to monitor temperature variations. If the temperature is varying, independently-controlled air-conditioning will be needed to keep the room temperature constant (though note the section on ‚"air currents" below)
• Allow the microscope to warm up. Turn on all electronics and lamps at least 4-5 hours before beginning a timelapse acquisition. Experiment with your system by changing one thing at a time and monitoring how that factor affects drift. For instance, it may be necessary to leave incubation units and the microscope electronics on permanently, but switch on HBO or other fluorescence sources only 1 hour before starting imaging.

Controlling the thermal environment of your microscope is essential for maintaining long-term focus and XY stability. The sensitivity of your experiments to drift will depend on the objective magnification that is used - higher magnifications will be more sensitive to drift and will require longer warm-up times.

We recommend that users experiment with different "warm-up" regimes and choose one that brings drift to within acceptable levels for their experiment.

Air currents and vibration acting on the slide or sample can cause drift. Symptoms of air-current drift may include:

• Seemingly random shifts in focus
• Seemingly random shifts in XY position

Ensure that:

• Air-conditioning and fans do not blow directly onto the microscope
• Opening and closing of doors does not cause sudden pressure changes in the room. Dampen doors if necessary to stop them opening or closing quickly.
• The microscope is sufficiently isolated from vibration and shocks using an anti-vibration or air table.

It is possible that an XY stage or Z controller is driving an axis very slowly. Turn off all the components and quantify the drift again each time. If the drift stops when a component is off, but returns when it is switched on, it is likely that this component is at fault.

To solve this:

• If the XY stage comes with a joystick, switch the stage controller off, unplug the joystick, switch the stage controller on again and repeat the drift test. If the drift stops, it is possible that the joystick is not centering properly. Contact the helpdesk for assistance.
• Any other problem may be a hardware fault. Contact the helpdesk for assistance.